94
T T H H È È S S E E En vue de l'obtention du DOCTORAT DE LUNIVERSITÉ DE TOULOUSE Délivré par l'Université Toulouse III - Paul Sabatier Discipline ou spécialité : Microbiologie et Génétique moléculaire JURY Dr Arturo MUGA, Professeur, Universidad del Pais Vasco, Espagne Dr Alessandra POLISSI, Professeur, Università degli Studi di Milano-Bicocca, Italie Dr Peter FALLER, Professeur, Laboratoire de Chimie de Coordination, Toulouse Dr Jan Willem DE GIER, Professeur associé, Stockholm University, Suède Dr Marie-Pierre CASTANIE-CORNET, Maître de Conférence, Université de Toulouse Dr Pierre GENEVAUX, Directeur de Recherche, CNRS, Toulouse Ecole doctorale : Biologie, Santé, Biotechnologie (BSB) Unité de recherche : Laboratoire de Microbiologie et Génétique Moléculaire (LMGM) Directeur(s) de Thèse : Dr Pierre GENEVAUX (Directeur), Dr Marie-Pierre CASTANIE-CORNET (co- Directeur) Membre invité: Dr Joen LUIRINK (co-Superviseur) Rapporteurs : Dr Arturo MUGA, Dr Alessandra POLISSI Présentée et soutenue par Nicolas Bruel Le Mardi 2 avril 2013 Titre : Hsp33 controls Elongation Factor-Tu stability and allows Escherichia coli growth in the absence of the major DnaK and TriggerFactor chaperones

T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

  • Upload
    others

  • View
    1

  • Download
    0

Embed Size (px)

Citation preview

Page 1: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par l'Université Toulouse III - Paul Sabatier

Discipline ou spécialité : Microbiologie et Génétique moléculaire

JURY

Dr Arturo MUGA, Professeur, Universidad del Pais Vasco, Espagne

Dr Alessandra POLISSI, Professeur, Università degli Studi di Milano-Bicocca, Italie

Dr Peter FALLER, Professeur, Laboratoire de Chimie de Coordination, Toulouse

Dr Jan Willem DE GIER, Professeur associé, Stockholm University, Suède

Dr Marie-Pierre CASTANIE-CORNET, Maître de Conférence, Université de Toulouse

Dr Pierre GENEVAUX, Directeur de Recherche, CNRS, Toulouse

Ecole doctorale : Biologie, Santé, Biotechnologie (BSB)

Unité de recherche : Laboratoire de Microbiologie et Génétique Moléculaire (LMGM)

Directeur(s) de Thèse : Dr Pierre GENEVAUX (Directeur), Dr Marie-Pierre CASTANIE-CORNET (co-

Directeur)

Membre invité: Dr Joen LUIRINK (co-Superviseur)

Rapporteurs : Dr Arturo MUGA, Dr Alessandra POLISSI

Présentée et soutenue par Nicolas Bruel Le Mardi 2 avril 2013

Titre : Hsp33 controls Elongation Factor-Tu stability and allows Escherichia coli growth in

the absence of the major DnaK and TriggerFactor chaperones

Page 2: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such
Page 3: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

2

Contents

ABBREVIATIONS .............................................................................................................................. 5

RÉSUMÉ .............................................................................................................................................. 6

SUMMARY ......................................................................................................................................... 7

INTRODUCTION ................................................................................................................................ 9

Part I: Protein folding and molecular chaperone functions .............................................................. 9

1- Protein folding within the cell ............................................................................................. 9

2- Role of the mRNA and the translation machinery ......................................................... 10

a- Rare codons, translational pausing and protein folding ................................................. 10

b- tRNA concentration modulates nascent chain protein folding ........................................ 11

c- Roles of EF-Tu ................................................................................................................. 12

d- Chaperone function of the ribosome ................................................................................ 13

e- Molecular chaperones, concept, functions and universality............................................ 14

Part II: Chaperone-assisted de novo protein folding ...................................................................... 15

1- The ribosome-bound Trigger Factor ............................................................................... 15

a- Structure / interaction with the ribosome ........................................................................ 15

b- TF substrate interaction and cellular functions ............................................................... 19

2- DnaKJE cycle and interactors .......................................................................................... 21

a- DnaKJE structure and cycle ............................................................................................ 21

b- Functions of DnaK ........................................................................................................... 24

c- Interactors in the cell ....................................................................................................... 24

3- The chaperonin GroESL ................................................................................................... 25

a- Structure of the GroESL complex and chaperonin cycle ................................................. 25

b- Interactors in the cell ....................................................................................................... 26

4- Interplay between TF/DnaKJE/GroESL during de novo protein folding .................... 27

Part III: Interplays between TF, DnaKJE and GroESL and other chaperones during protein

export .............................................................................................................................................. 29

1- Sec Translocation system .................................................................................................. 30

2- Twin Arginin translocation system .................................................................................. 31

Part IV: Chaperone-mediated response to protein misfolding and aggregation ............................. 33

1- Molecular chaperone networks and protein disaggregation ......................................... 33

a- Presentation of other chaperones involved in protein disaggregation ............................ 33

b- Chaperone networks involved in protein disaggregation ................................................ 34

Page 4: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

3

2- Hsp33 as a member of the chaperone network ............................................................... 37

a- Hsp33 is involved in the oxidative stress response .......................................................... 37

b- Structure of Hsp33 ........................................................................................................... 38

c- Activation cycle of Hsp33 ................................................................................................ 39

d- Hsp33 deletion and functions in the cell .......................................................................... 42

MATERIALS AND METHODS ....................................................................................................... 44

1- Strains and plasmids ......................................................................................................... 44

a- Bacterial Strains, phages, and culture conditions ........................................................... 44

b- Plasmid construction ....................................................................................................... 45

2- In vivo experiments ............................................................................................................ 46

a- Bacterial viability assay and genetic experiments ........................................................... 46

b- Isolation of protein aggregates and cell fractionation .................................................... 46

c- In vivo pull-down assay ................................................................................................... 47

3- In vitro experiments ........................................................................................................... 47

a- Western blot analysis ....................................................................................................... 47

b- In vitro translation and cross-linking experiments .......................................................... 48

c- Pulse-chase and immunoprecipitation analyses .............................................................. 48

RESULTS ........................................................................................................................................... 50

1- Hsp33 overproduction supports bacterial growth and prevents protein

aggregation in the absence of TF and DnaK .......................................................................... 50

2- Hsp33 function is critical in the absence of both TF and DnaK .................................... 54

3- Hsp33 specifically interacts with EF-Tu .......................................................................... 58

4- Hsp33 triggers EF-Tu degradation by the stress protease Lon ..................................... 63

5- EF-Tu inhibition helps bacterial growth in the absence of TF and DnaK ................... 65

DISCUSSION .................................................................................................................................... 69

REFERENCES ................................................................................................................................... 74

REMERCIEMENTS .......................................................................................................................... 93

Page 5: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

4

Page 6: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

5

ABBREVIATIONS

AAA+: ATPases Associated with a variety of cellular Activities

DNA: DesoxyriboNucleic Acid

EF-Tu: Elongation Factor-Tu

EF-Ts: Elongation Factor-Ts

FRET: Fluorescence resonance energy transfer

HSP: Heat Shock Protein

IPTG: isopropyl β-D-1-thiogalactopyranoside

JDP: J-domain protein

kDa: KiloDalton

NAC: Nascent polypeptide-Associated Complex

NBD: Nucleotide Binding Domain

NEF: Nucleotide Exchange Factor

NMR: Nuclear Magnetic Resonance

OMP: Outer-Membrane β-barrel Protein

PPIase: Peptidyl-Prolyl cis/trans Iisomerase

RAC: Ribosome-Associated Complex

REMP : Redox Enzyme Maturation Protein

RNA: RiboNucleic Acid

SBD: Substrate Binding Domain

sHSP: small Heat Shock Protein

SRP: Sgnal Recognition Particle

Tat: Twin-Arginine Translocon

TF: Trigger Factor

Page 7: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

6

RÉSUMÉ

Le repliement intracellulaire des protéines nouvellement synthétisées est assisté par

des réseaux cellulaires de protéines chaperons. Chez Escherichia coli, la coopération entre les

protéines chaperons Trigger Factor (TF) et DnaK est prédominante dans ce processus. En

accord avec ceci, la délétion simultanée des gènes codants pour ces deux protéines chaperons

conduit à une croissance bactérienne très réduite et à l’accumulationd’un grand nombre de

protéines cytoplasmiques sous forme d’agrégats. Au cours de cette étude, nous avons utilisé

ces phénotypes afin de mettre en évidence des interactions potentielles au sein du réseau de

protéines chaperons in vivo. Nous avons montré que la perte des protéines chaperons TF et

DnaK, et donc des voies de repliements dans lesquelles elles sont impliquées, pouvait être

secourue de façon efficace par la surexpression du chaperon Hsp33, connu pour être activable

en réponse à un stress oxydatif sévère. En outre, la délétion du gène hslO, codant pour Hsp33,

n’était plus tolérée en l’absence de TF et DnaK. Cependant, en comparaison avec d’autres

protéines chaperons comme GroESL ou SecB, la suppression de ces phénotypes par Hsp33

n’a pas pu être attribuée à un éventuel chevauchement de fonctions avec DnaK et TF. Au

contraire, nos résultats montraient qu’ Hsp33 surexprimée fixait de façon spécifique le facteur

d’élongation-Tu (EF-Tu) et favorisait sa dégradation par la protéase Lon. Cette action

synergétique entre Hsp33 et Lon était responsable du rétablissement de la croissance

bactérienne en l’absence de TF et DnaK, possiblement via le rétablissement du couplage entre

la vitesse de traduction et les capacités de repliement des protéines nouvellement synthétisées

du double mutant. Afin de soutenir cette hypothèse, nous avons ensuite montré que la

surexpression de la toxine HipA qui inhibe EF-Tu, était aussi capable de supprimer le

phénotype de thermosensibilité et de réduire significativement l’agrégation des protéines en

l’absence de TF et DnaK.

Page 8: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

7

SUMMARY

Intracellular de novo protein folding is assisted by cellularnetworks of molecular

chaperones. In Escherichia coli, cooperationbetween the chaperones Trigger Factor (TF) and

DnaK iscentral to this process. Accordingly, the simultaneous deletion of both chaperone-

encoding genes leads to severe growth andprotein folding defects. Herein, we took advantage

of suchdefective phenotypes to further elucidate the interactions ofchaperone networks in

vivo. We show that disruption of theTF/DnaK chaperone pathway is efficiently rescued by

over-expressionof the redox-regulated chaperone Hsp33. Consistentwith this observation, the

deletion of hslO, the Hsp33 structuralgene, is no longer tolerated in the absence of the

TF/DnaK pathway.However, in contrast with other chaperones like GroESL orSecB,

suppression by Hsp33 was not attributed to its potentialoverlapping general chaperone

function(s). Instead, we showthat over-expressed Hsp33 specifically binds to elongation

factor-Tu (EF-Tu) and targets it for degradation by the proteaseLon. This synergistic action of

Hsp33 and Lon was responsiblefor the rescue of bacterial growth in the absence ofTFand

DnaK,by presumably restoring the coupling between translation andthe downstream folding

capacity of the cell. In support of thishypothesis, we show that over-expression of the stress-

responsivetoxin HipA, which inhibits EF-Tu, also rescues bacterialgrowth and protein folding

in the absence of TF and DnaK.

Page 9: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

8

INTRODUCTION

Page 10: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

9

INTRODUCTION

Part I: Protein folding and molecular chaperone functions

1- Protein folding within the cell

Proteins need to reach their native three-dimensional structure to become active. This

folding process is determined by the primary amino-acid sequence (Anfinsen, 1973). Protein

folding is not a random event but follows a specific energetic landscape through which

protein gets some native-like structure as folding intermediate. These numerous pathways

drive the protein to its native structure at the most stable energetic point (Dobson, 2004;

Onuchic & Wolynes, 2004). Even if small single domain proteins, having less than 100

residues, can reach their native state without such intermediates (Jackson, 1998), folding of

multi-domain proteins generally need parallel routes of “nucleation-condensation” of a small

number of key residues, which allow the protein to condensate up to its native structure

(Radford et al, 1992). Within the cell, such folding events coupled to the crowded cellular

environment (i.e. proteins, nucleic-acids, complex sugars in high concentration ~400g/L (Ellis

& Minton, 2003)) can lead to protein misfolding and aggregation, often associated with

human diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract

such noxious pathways, intricate quality control systems have been maintained in all

kingdoms of life (Hartl & Hayer-Hartl, 2002). Such quality controls initially take place during

translation of nascent peptide chains by direct action on the ribosome and/or translational

factors such as tRNAs, the elongation factor Tu (EF-Tu) and the mRNA itself (Caldas et al,

1998; Fedyunin et al, 2012; Komar, 2009; Voisset et al, 2008). Other factors known as

“Molecular Chaperones” actively participate in this folding process by interacting directly

with polypeptide chains in a co- and/or post-translational manner to prevent their aggregation

(Fig. 1).

Page 11: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

10

2- Role of the mRNA and the translation machinery

a- Rare codons, translational pausing and protein folding

The folding of nascent peptide chain is incorporated in the kinetics of mRNA

translation during its elongation (Komar, 2009; Komar et al, 1999; Krasheninnikov et al,

1991; Wolin & Walter, 1988). In fact, mRNA sequences contain regions composed of rare

codons slowing down the rate of translation and influencing the folding of the nascent peptide

chain during its translation. Presence of these rare codons is due to the distribution of

aminoacyl-tRNA isoacceptor species in a given organism (Chavancy & Garel, 1981; Garel,

1974; Ikemura, 1981). Such a variation in aminoacyl-tRNA isoacceptor concentration creates

a strong codon bias within any given organism (Sharp et al, 1988). Pause sites were identified

Chaperones

Nascent polypeptide Intermediate Native protein

Aggregates

Fibrillar Amyloids

Fig. 1: Folding pathways of a newly synthesized protein. Proteins can pass through folding intermediates

before reaching their native structure. Misfolding events such as aggregation may occur at each folding step.

Molecular chaperones prevent this aggregation and favor the folding of nascent polypeptides until their native

conformation.

Page 12: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

11

by micrococcal nuclease protection assay of mRNA during translation (Hollingsworth et al,

1998; Wolin & Walter, 1988). Indeed, a frequently used codon, which corresponds to a high

concentration in its aminoacyl-tRNA isoacceptor, is translated faster than one infrequently

used. So that kind of punctuation into the mRNA during the translation facilitates, to some

extent, the co-translational folding of the nascent polypeptide because of the positioning of

these pause sites at specific domains in the encoded protein. Remarkably, a correlation

between codon abundance and domain boundaries has been identified (Purvis et al, 1987). In

addition, it has been found that α-helices are frequently associated with high-frequency

codons, whereas β-strands and random coils are preferentially coded by rare codon regions

(Thanaraj & Argos, 1996). Moreover, the location of these pause sites is highly conserved in

some protein families including cytochromes C, globins, γ-B crystallins and chloramphenicol

acetyltransferase (Krasheninnikov et al, 1989; Widmann et al, 2008). To study the relevance

of such pause sites, silent mutations of mRNAs coding the Escherichia coli chloramphenicol

acetyltransferase, Saccharomyces cerevisiae anthranilate synthase indole-3-glycerol

phosphate synthase and Echinococcus granulosus fatty acid binding protein, replacing rare

codons by common ones showed a decrease in their specific activities when compared to the

wild type proteins (Cortazzo et al, 2002; Crombie et al, 1994; Komar et al, 1999).

Furthermore, the proteins produced from these silently mutated mRNAs were misfolded.

Although the kinetics of translation appear to play a significant role in the co-translational

folding of nascent peptide chain, but there are additional factors, including tRNA

concentration, Elongation Factor-Tu (EF-Tu), and ribosome, which participate directly or

indirectly to the folding process.

b- tRNA concentration modulates nascent chain protein folding

tRNAs bring amino-acids to their cognate codons. Remarkably, it has been shown

both in vitro and in vivo that the concentration of tRNAs acceptors can drastically modulate

the rate of translation. Indeed, using an in vitro translational experiment, Anderson first

showed that translation of a poly-U by tRNAPhe

was inhibited at high concentration of

tRNAPhe

(tRNAPhe

/70S ribosome ratio of 7) (Anderson, 1969). In addition, a different study

pointed out that in the presence of a constant concentration of exogenous tryptophan, the rate

of incorporation of this amino acid was considerably diminished when tRNATrp

concentration

was too high, following over-expression of tRNATrp

from a plasmid. Such over-expression

changed the ratio of uncharged tRNATrp

/ [Trp-tRNATrp

] and the pool of uncharged tRNATrp

was found to inhibit Trp incorporation into proteins (Rojiani et al, 1990). Remarkably, they

Page 13: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

12

also showed that uncharged tRNATrp

could inhibit incorporation of other amino acids, such as

Lys and Cys (Rojiani et al, 1990). A third team studied the effect of low-abundant tRNAs

upregulation on the folding and solubility of E. coli proteins (Fedyunin et al, 2012). In this

case, upregulation of argU which encodes tRNA4Arg

that pairs with arginine codons AGA and

AGG, ileY encoding for tRNA2Ile

that pairs with AUA codon or leuW encoding tRNA3Leu

that

pairs with CUA codon, modifying the rate of translation of these rare condons, was

responsible of the aggregation of many proteins such as molecular chaperones which had been

found to suffer from this misfolding effect (Fedyunin et al, 2012). This approach revealed the

importance of the translational rate during co-translational folding of all the cellular proteome

and the role of tRNA isoacceptors concentration in this translational rate.

c- Roles of EF-Tu

In E. coli, EF-Tu is the most abundant protein in the cell, representing 5 to 10% of the

total proteins (Furano, 1975). EF-Tu has a GTPase activity and brings aminoacyl-tRNA to the

A site of the ribosome in a GTP-dependent manner. GDP is replaced through interaction with

the nucleotide exchange factor EF-Ts (Agirrezabala & Frank, 2009). EF-Tu and its eukaryotic

homolog EF-1α have many other cellular functions. EF-Tu was found to be an essential

subunit of the Qβ phage replicative complex (Blumenthal et al, 1972). EF-Tu can also

stimulate RNA synthesis by interacting with the transcriptional apparatus (Travers et al,

1970). In Bacillus subtilis, EF-Tu plays an additional role in cell shape maintenance,

interacting with the actin-like MreB (Defeu Soufo et al, 2010). This role was also identified

for EF-1α, as it can bind actin filaments and microtubules and influence the assembly and the

stability of these cytoskeletal polymers (Shiina et al, 1994; Yang et al, 1990). In addition,

Caldas and coworkers also found that EF-Tu could reactivate chemically-unfolded citrate

synthase and α-glucosidase with the same efficiency than DnaKJE in vitro, thus suggesting

chaperone function (Caldas et al, 1998). The eukaryotic EF-1α chaperone activity was also

identified using Phenylalanyl-tRNA synthase and seryl-tRNA synthase as substrates. In this

case, it was shown that the mammalian EF-1α (called eEFiA) could refold and restore the

enzymatic activity of these two aminoacyl-tRNA synthase (Lukash et al, 2004). In conclusion,

EF-Tu does not only bring aminoacyl-tRNAs to the ribosome A site but participates in a

plethora of cellular processes, most likely including protein folding. The last compound of the

translational machinery to have a chaperone function protecting and actively participating in

the co-translational folding of the nascent chain protein is the ribosome itself.

Page 14: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

13

d- Chaperone function of the ribosome

Ribosomes represent 30% of the total cell mass with up to 105 and 10

6 ribosomes in a

bacteria and a mammalian cell respectively (Bashan & Yonath, 2008). It is a

ribonucleoprotein complex composed of 2 subunits. The small subunit (30S in bacteria; 40S

in eukaryote) decodes the mRNA during translation and the large subunit (50S in bacteria;

60S in eukaryote) contains the peptidyl transferase center and the ribosomal exit tunnel

through which nascent polypeptides will emerge and be released into the cytosol. The length

of the exit tunnel is about 80 Å to 100 Å with a diameter comprised between 10 Å at its

narrowest point and 20 Å at its widest point. (Ban et al, 2000; Harms et al, 2001; Nissen et al,

2000). These dimensions allow the ribosome to contain a nascent chain of 30 amino-acids in

an extended conformation or 60 amino-acids as an α-helix (Malkin & Rich, 1967; Picking et

al, 1992; Voss et al, 2006). During translation, nascent chain interacts with this exit tunnel

composed of 23S rRNA and proteins L4, L22 and L23 in bacteria (Ban et al, 2000; Harms et

al, 2001; Kosolapov & Deutsch, 2009; Lu & Deutsch, 2005; Lu et al, 2007; Nissen et al,

2000). Such interactions can regulate kinetics of translation and induce translational pausing,

thus facilitating co-translational folding of the nascent peptide chain or its interaction with

ribosome associated factors such as the molecular chaperone Trigger Factor in bacteria (TF)

or the signal recognition particle (SRP) involved in the co-translational targeting of secretory

or membrane proteins to the sec translocon (Eisner et al, 2006; Kramer et al, 2009; Marin,

2008). Moreover, the ribosomal exit tunnel directly participates to the folding of nascent

chains by promoting its compaction or its helical conformation in the lower and upper part of

the tunnel. The middle part of the tunnel is constricted by L4 and L22 proteins (Lu &

Deutsch, 2005). This constriction site avoids protein folding in this area and, interacting

directly with the nascent chain, may modulate the docking site shared by TF or SRP and then

allow the recruitment of each of them to the ribosome exit tunnel (Gu et al, 2003; Houben et

al, 2005; Ullers et al, 2003). The exit pore of the tunnel also plays a role in the co-translational

folding of the nascent chain allowing the formation of tertiary structures such as β-hairspins

(Kosolapov & Deutsch, 2009).

The ribosome itself also possesses some chaperone activity. Indeed, the 50S ribosomal

subunit can interact post-translationally with unfolded proteins and efficiently participate to

their folding (Kudlicki et al, 1994; Voisset et al, 2008). The interaction site of the ribosome

with unfolded protein had been identified near from the peptidyl transferase center, in the

domain V of the 23S rRNA (Chattopadhyay et al, 1996; Pal et al, 1997). The ribosome can

Page 15: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

14

also participate in the co-translational folding of newly synthesized proteins in a different

way. Indeed, Kaiser and coworkers (2011) studied the active folding activity of the ribosome

exit tunnel following the folding of the T4 lysozyme and found that the ribosome could

prevent misfolding of the nascent chain by modulating its folding rate (Kaiser et al, 2011).

Furthermore, this exit pore is a docking site for many key proteins involved in the enzymatic

processing of the nascent chain such as the methionine aminopeptidase and the peptide

deformylase (Kramer et al, 2009), for targeting factors such as SRP and SecA, ribosome

associated chaperones like Trigger Factor in bacteria or Ssb/Ssz/Zuotin chaperone triad,

ribosome-associated complex (RAC) and nascent polypeptide-associated complex (NAC) for

the eukaryote S. cerevisiae(Eisner et al, 2006; Ferbitz et al, 2004; Preissler & Deuerling,

2012).

e- Molecular chaperones, concept, functions and universality

Molecular chaperones facilitate protein folding and prevent and/or rescue noxious

protein aggregation. They are involved in many cellular pathways such as: de novo protein

folding, protein disaggregation, protein translocation through a biological membrane,

oligomerisation and control of protein-protein interaction, as well as protein turn over

(Hendrick & Hartl, 1993). Molecular chaperones are conserved in all the three kingdoms of

life, represented by different protein families, and are known to function in a sequential

manner on their substrates co-translationally and post-translationally (Frydman et al, 1994;

Langer et al, 1992; Siegers et al, 1999).

A first group of molecular chaperones interacts with the ribosome and/or the nascent

chain to facilitate co-translational folding. Bacteria use the chaperone Trigger Factor,

interacting at the same time with the ribosome and the nascent chain (Hesterkamp et al, 1996).

The Hsp70 chaperone DnaK from E. coli can also interact with nascent polypeptides to assist

them during their co-translational folding (Teter et al, 1999). Although the eukaryotic

chaperone system does not have Trigger Factor, they do have two different kinds of ribosome

associated complexes acting at the ribosome exit pore to support co-translational folding

(Preissler & Deuerling, 2012). Indeed, in Saccharomyces cerevisiae, the ribosome-associated

complex (RAC) composed of the Hsp70/Hsp40 chaperone system Ssz/Zuotin and another

Hsp70 named Ssb and the heterodimeric nascent chain-associated complex (NAC) may

parallel Trigger Factor function in the folding of the nascent polypeptide chains (Preissler &

Deuerling, 2012).Hsp70 may also bind substrates in a post-translational way to mediate their

folding (Deuerling et al, 1999; Teter et al, 1999). The chaperonins represent the second group

Page 16: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

15

of molecular chaperones, mostly participating in the post-translational folding of proteins.

They are structured as double-ring complexes forming a central cavity in each ring, binding

and encapsulating unfolded proteins in one ring, which protect the substrate from the crowded

environment of the cell and help it to fold (Mayhew et al, 1996; Weissman et al, 1996). Other

types of chaperones are more specifically involved in disaggregation, as it’s the case for

Hsp90 and Hsp100, HtpG and ClpB in E. colirespectively.

In the following parts of the introduction, the structures, the mechanisms and the

functions of the major E. coli chaperones involved in de novo protein folding, namely TF,

DnaKJE and GroESL, will be presented. In addition, their functional cooperation with other

chaperone systems during protein export, protein disaggregation and in response to severe

oxidative stress will be developed.

Part II: Chaperone-assisted de novo protein folding

1- The ribosome-bound Trigger Factor

a- Structure / interaction with the ribosome

The ribosome-bound TF is the first molecular chaperone to interact with most of the

newly synthesized polypeptides (Valent et al, 1995). It is a 48kDa protein constituted of 3

distinct domains elongated in a Dragon-like shaped structure (Ferbitz et al, 2004; Hesterkamp

& Bukau, 1996; Zarnt et al, 1997) (Fig. 2A). The N-terminal domain of TF, composed of

amino acids 1 to 149, is the ribosome binding domain, which also contributes to TF chaperone

activity. This domain possesses some structural homology with the molecular chaperone

Hsp33 except for the additional loop involved in the binding of TF to the ribosome via the TF

signature motif “GFRxGxxP” (Genevaux et al, 2004; Hoffmann et al, 2010; Kramer et al,

2002; Kristensen & Gajhede, 2003). A long linker (aa 111 to 149) is found between the

ribosome binding domain and the peptidyl-prolyl cis/trans isomerase domain (PPIase) of TF.

The PPIase domain (aa 150 to 245) is related to the FK506 binding protein family of PPIase

(Hesterkamp & Bukau, 1996; Scholz et al, 1997; Stoller et al, 1995). This domain is

dispensable for chaperone function of TF in vivo, but it can enhance chaperone activity of TF

as an auxiliary binding site of substrates (Genevaux et al, 2004; Hoffmann et al, 2006; Kramer

et al, 2004a; Lakshmipathy et al, 2007; Merz et al, 2006). The C-terminal domain (aa 246 to

432), forming the body of the dragon, is found between the N-terminal domain (the tail of the

Page 17: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

16

dragon) and the PPIase domain (the head of the dragon). It interacts with the long linker (aa

111 to 149), which stabilize its structure containing 2 protruding helical “arms” (Ferbitz et al,

2004; Martinez-Hackert & Hendrickson, 2007; Merz et al, 2006). This domain forms the main

chaperone module of TF (Merz et al, 2006).

TF is conserved in bacteria and chloroplasts, but its PPIase domain may be absent in

some species (Hoffmann et al, 2010; Kristensen & Gajhede, 2003). Some bacteria, such as

Desulfitobacterium hafniense, even possess several TF and TF-like molecular chaperones

with specific functions (Maillard et al, 2011). Indeed, among the 3 TF homologs present in

this bacterium, the TF-like chaperone PceT, which lacks the TF N-terminal domain, is

dedicated to the folding and the transport of the reductive dehalogenase PceA through the

Twin arginine translocation system (Maillard et al, 2011).

149 245 432360

NC

Ribosome

binding

domainPPIase

domain Arm 1 Arm 2

A B

43 50

GFRxGxxP

Fig. 2: Structure of the E. coli TF and interaction with ribosome exit tunnel. (A) The upper

part represents the three-dimensional structure ribbon diagram of TF (PBD 1W26). The bottom

shows the domain arrangement of TF and the TF signature motif of the ribosome binding site (aa

43 to 50). (B) Interaction of TF with the exit-tunnel of the ribosome. TF binds L23 and L29 and

covers the exit tunnel from the crowded environment. The star represents the exit pore of the

tunnel. Ribosome binding site of TF is represented in red, the PPIase domain is in yellow, the

Arm 1 is in green and the Arm 2 in blue. Figure adapted from (Ferbitz et al, 2004).

Page 18: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

17

TF is an abundant cytosolic protein represented in 2- to 3- fold molar excess over

ribosome (50µM in spite of 20µM respectively) (Crooke et al, 1988). It is constitutively

expressed and dispensable under normal growth conditions (Martinez-Hackert &

Hendrickson, 2009). TF cycles on and off the ribosome in 1:1 stoichiometry in an ATP-

independent manner and creates a protective arch over the ribosomal exit tunnel (Fig. 2B)

(Hoffmann et al, 2010; Kaiser et al, 2006; Rutkowska et al, 2008). During the TF

binding/release cycle, it has been found that the presence of a nascent chain increases the

affinity of TF for the ribosome 2 to 30 fold (Raine et al, 2006; Rutkowska et al, 2008). After

release from the ribosome, TF may stay bound to the nascent chain of large multi-domain

proteins allowing another free TF to bind the ribosome and in some cases to facilitate transfer

of its substrate to the downstream chaperones DnaKJE and GroESL (Kaiser et al, 2006;

Lakshmipathy et al, 2010). In addition, it has been found that TF from Thermotoga maritima

TF (tmTF) can dimerize and encapsulate 2 tmS7 ribosomal proteins (Martinez-Hackert &

Hendrickson, 2009). Such a dimerization of tmTF could create an Anfinsen-like cage which

could protect S7 and participate to its incorporation into the ribosome (Martinez-Hackert &

Hendrickson, 2009) (Fig. 3).

The N-terminal of TF interacts with the ribosome via its signature motif to the L23

ribosomal protein located at the exit port, the ribosomal RNA 23S and additional interaction

occurs with the L29 ribosomal protein (Baram et al, 2005; Kramer et al, 2002; Schlunzen et

al, 2005) (Fig. 2B). This TF-ribosome interaction is crucial for TF chaperone function during

co-translational folding of nascent polypeptide chains (Lakshmipathy et al, 2007). In vitro

experiments showed an early interaction with nascent polypeptide chains as short as 40 amino

acids (Merz et al, 2008). Nevertheless, using ribosome profiling of purified ribosome –

nascent chain – TF complexes and deep sequencing of mRNA fragments protected by these

ribosomes, Oh and coworkers (2011) showed that, in vivo, TF seems to preferentially bind to

nascent polypeptide chains of about 100 amino acids (Oh et al, 2011). This late interaction

between TF and the nascent chain potentially allows earlier interaction with other ribosome

associated nascent chain interacting factors, including the peptide deformylase, the

methionine aminopeptidase and SRP (Ball & Kaesberg, 1973; Bingel-Erlenmeyer et al, 2008;

Keenan et al, 2001).

Recent works identified nascent chains that are disliked by TF: a segment of poly-

alanine folded into a helical conformation (Bhushan et al, 2010; Lu & Deutsch, 2005). Indeed,

replacing 9 amino acids in the N-terminal of GFP by a poly-Alanine sequence near the exit

Page 19: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

18

tunnel, the center of the tunnel or the peptidyl transferase center place this helical structure in

different parts of the ribosome tunnel. Using this technique, Lin and coworkers showed that

TF binding to the ribosome was disfavored when this helical structure was in an area near

from the exit tunnel and interacted with L23. This interaction can induce conformational

changes in the ribosomal binding site of TF and reduce its recruitment to the exit pore (Lin et

al, 2012). To validate this approach, they used the signal anchor sequence of a cytoplasmic

membrane protein and the signal sequence of the secretory protein pre-β-lactamase

(Bornemann et al, 2008). These sequences are known to fold into the ribosome tunnel in a

helical structure and to enhance the recruitment of SRP (Berndt et al, 2009; Bornemann et al,

2008; Halic et al, 2006; Woolhead et al, 2004). Both of them reduced the recruitment of TF at

the ribosome exit tunnel after interaction with L23 inside the ribosomal tunnel (Lin et al,

2012). Therefore, the recruitment of TF to the exit pore is modulated by the nascent peptide

chain itself, which modifies the ribosome binding site shared by TF and SRP (Lin et al, 2012).

Trigger Factor

50S

L23

50S

L23

Native proteinProtein

complex

Free dimer

of TF

Fig. 3: Chaperone cycle and assembly function of TF. TF interacts with nascent chains by direct

association with the ribosome. More than one TF molecule may bind a nascent polypeptide of a multi-

domain protein. This protein can be released in a native state or TF may stay bound to it in the cytosol to

fulfill its folding. Martinez-Hackert and Hendrickson (2009) also identified a new role of TF in the assembly

of protein complexes. TF could also form dimers and encapsulate two substrates mediating their folding and

their interaction. This role is important for ribosome biogenesis.

Page 20: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

19

b- TF substrate interaction and cellular functions

Several studies were developed to identify preferred interactors of TF. A screening of

2.842 membrane coupled peptides of 20 different proteins originating from bacteria and

eukaryotes was used to identify TF binding motifs (Patzelt et al, 2001). The analysis revealed

that TF preferentially interacts with motifs of 8 residues enriched in aromatic residues (Phe,

Tyr, Trp and His) and basic residues (Arg and Lys) and impoverished in acidic residues (Asp

and Glu). Even if TF possesses a PPIase activity, proline residues do not seem to participate

in substrate binding. TF motifs occurs frequently, about every 32 residues in all protein tested.

This frequent motif allows TF to interact virtually with all the proteins during translation

(Patzelt et al, 2001). However, interactome studies pointed out some preferential substrates of

TF. A comparison between co-purified TF substrates and aggregated proteins isolated in a tig

null-mutant identified 178 potential TF substrates, a majority of them being ribosomal

proteins or proteins from homo- or hetero-oligomers (Martinez-Hackert & Hendrickson,

2009). Recently, a quantitative ribosome profiling analysis of ribosomes whose nascent chains

are bound to TF isolated outer-membrane β-barrel proteins (OMPs) as the strongest TF

interactors (Oh et al, 2011).

Even if TF interacts with a large number of substrates, the mode of action may differ.

TF can help peptide domains shorter than 150 residues to fold within its cradle (Hoffmann et

al, 2006; Merz et al, 2008). In vitro experiments also showed that TF promotes folding of

denatured proteins (Huang et al, 2000; Kramer et al, 2004a; Merz et al, 2006). In addition, TF

can delay co-translational folding of multi-domain proteins like firefly luciferase and β-

galactosidase as presented in the cycle of TF (Part II 1-a). This conclusion has been reached

by measuring the time-dependent enzymatic activity of these two proteins with or without TF

(Agashe et al, 2004). Two recent works have further studied such TF function. Indeed,

Hoffmann and colleagues (2012) showed that TF was found to postpone the disulfide bond

formation of ribosome arrested β-lactamase and barnase, to unfold a folded arrested barnase

and to facilitate its degradation. In addition, modeling the co-translational folding of the N-

terminal domain of β-galactosidase (216 residues), O’Brien and coworkers proposed that TF

could act on the nascent peptide via three different mechanisms: TF might (i) decrease the rate

of structural rearrangements, (ii) avoid tertiary structure formation, (iii) increase the effective

length of the exit tunnel and entanglements between the nascent polypeptide chains (O'Brien

et al, 2012). Such functions of TF could significantly help multi-domain proteins to fold

correctly, thus avoiding protein misfolding.

Page 21: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

20

Moreover, interaction of the ribosome-bound TF with its substrate can facilitate

substrate degradation by proteases under certain conditions. Indeed, in vitro experiments

showed that, in the absence of DnaK, TF could favor the degradation of the arrested complex

barnase-arrested/ribosome (Hoffmann et al, 2012). In vivo works also revealed that TF favors

the degradation of EF-Tu in the absence of DnaK following Hsp33 over-expression (Bruel et

al, 2012). These results may pin point a new role of TF when DnaK is sequestered on

aggregates during a stress.

TF may participate in OMPs stability as well (Crooke & Wickner, 1987; Oh et al,

2011; Ullers et al, 2007). Indeed, deletion of the tig gene encoding TF provokes a decrease in

the steady state levels of OMPs compared with wild type cells (Oh et al, 2011) and some

OMPs were isolated as strong TF interactors by ribosome profiling analysis (Oh et al, 2011).

However, in vivo experiments revealed that TF deletion does not facilitate export of SecB-

dependent presecretory proteins, including MBP, OmpA and SecA (Guthrie & Wickner,

1990; Lee & Bernstein, 2002; Ullers et al, 2007). Instead, the deletion of tig even accelerated

the export of some presecretory proteins with Sec translocon (Lee & Bernstein, 2002).

Another example of TF involved in protein secretion was identified in Streptococcus

pyogenes(Lyon & Caparon, 2003). TF is essential for the secretion and the maturation of the

cysteine protease SpeB. The PPIase activity of TF modifies SpeB then TF targets this

substrate to the Sec translocon (Lyon & Caparon, 2003). Moreover, TF over-expression

induces the aggregation of OmpF, partially reflecting the toxicity of TF (Genevaux et al,

2004). Finally, over-expression of TF is toxic and induces a strong filamentation phenotype

similar to the filamentation observed after FtsZ depletion (Genevaux et al, 2004; Guthrie &

Wickner, 1990; Ward & Lutkenhaus, 1985). These findings suggest that TF could be involved

in cell division. In some strain background, TF is also known to be essential for viability at

low temperature (10°C), perhaps due to its possible involvement in ribosome biogenesis and

cell division (Kandror & Goldberg, 1997).

In conclusion, TF facilitates the folding of nascent chains by protecting them from the

crowded cellular environment and delaying formation of unproductive folding intermediates.

In addition, it is involved in ribosome biogenesis and in the stabilization of OMPs.

Page 22: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

21

2- DnaKJE cycle and interactors

a- DnaKJE structure and cycle

The Hsp70 (Heat shock protein of 70kDa) molecular chaperone family is the most

conserved protein family in all organisms (Gupta, 1998). Thebest characterized Hsp70 family

member is the bacterial protein DnaK from E. coli. DnaK is a heat shock induced protein

present at about 10 000 copies per cell which double after a shift at 42°C (Genevaux et al,

2007). This molecular chaperone of 638 amino-acids in length possesses an N-terminal

nucleotide binding domain (NBD) of 45kDa with ATPase activity (aa 1 to 380) and a C-

terminal domain of 25kDa as substrate binding domain (SBD) subdivided into a β-sandwich

subdomain and a α-helical domain forming a lid (aa 398 to 638) (Flaherty et al, 1994). Both

domains are connected by a linker (aa 381 to 397) which is highly conserved and possesses a

characteristic 388DVLLLD393 hydrophobic segment essential for allosteric communication

between the NBD and the SBD (Mayer & Bukau, 2005; Swain et al, 2007; Vogel et al, 2006a;

Vogel et al, 2006b). In some eukaryotes the SBD contains an additional short motif

interacting with other partner proteins, like Hop/p60/CHIP that modulate chaperone activity

of Hsp70 (Mayer & Bukau, 2005; Young et al, 2004). The structure of a truncated DnaK (aa 1

to 605) bound to ADP and a substrate peptide (NRLLLTG) was revealed by NMR (Nuclear

Magnetic Resonance) presented in Fig. 4A (Bertelsen et al, 2009). This study showed that the

NBD and the SBD, in this conformation, are independent and mobile. This mobility is

restricted in a cone of ± 35° (Bertelsen et al, 2009). A recent work studied the structure of

DnaK bound to ATP. They showed that in this conformation, the α-helical lid and the β-

sandwich substrate pocket of the DnaK SBD were docked to different positions of the NBD

(Kityk et al, 2012). Then allosteric modifications induced by substrate binding and ATP

hydrolysis will unlock the SBD of DnaK (Kityk et al, 2012). DnaK facilitates protein folding

by cycles of binding/release of protein substrates. While ATP-bound DnaK has low affinity

and fast exchange rates for substrates, ADP-bound DnaK possesses a high affinity for the

substrate and a low exchange rate (Genevaux et al, 2007). The ATP hydrolysis by DnaK is

essential for its chaperone activity but this molecular chaperone has a low rate of ATP

hydrolysis (Karzai & McMacken, 1996; Laufen et al, 1999). Therefore, the DnaK chaperone

function relies on its obligate cochaperone partners, namely the J-domain protein Hsp40/DnaJ

and the nucleotide exchange factor GrpE (Schroder et al, 1993).

Page 23: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

22

The DnaKJE cycle starts with the interaction of a substrate-bound DnaJ dimer with the

ATP-bound DnaK. Indeed, DnaJ recruits the substrate and efficiently stimulates ATP

hydrolysis by DnaK up to 1000-fold (Karzai & McMacken, 1996; Laufen et al, 1999; Liberek

et al, 1991). ATP hydrolysis will close the SBD and enhance its affinity for the substrate.

Then, a second cochaperone, namely the nucleotide exchange factor GrpE will bind to the

NBD and facilitate ADP release. The subsequent binding of a new ATP opens the lid of the

SBD and stimulates substrate release (Harrison et al, 1997). Once released from DnaK, the

substrate can reach its native form or necessitates further DnaK cycles (Fig. 4B) or be

transferred to other chaperones, like the chaperonin GroESL to complete its folding (see part

II-4).

DnaK interacts with a broad range of substrates present in different conformations:

folded, misfolded or aggregated. DnaK was found to be highly efficient when the substrates

are misfolded but not aggregated. Indeed, a firefly luciferase variant from Photinus pyralis

was unfolded by Freeze-thaw cycles and urea and incubated in the presence of the DnaKJE

machinery (Sharma et al, 2010). Monitoring folding kinetics, the authors found that while

ATP hydrolysis induces substrate unfolding, substrate release induces spontaneous refolding

of the substrate (Sharma et al, 2010). Another study revealed that the lid of DnaK could stay

opened or partially opened even after hydrolysis of ATP (Kityk et al, 2012; Schlecht et al,

2011). This conformation could allow DnaK to interact with aggregated proteins or amyloids

and participate to their dissolution.

Page 24: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

23

NBD SBD

Linker

Lid

A

B

ATP

Pi

ADP

DnaK

DnaJ

Substrate

GrpE

ADP

ATP

ADP

+

++

Native

protein

ATP

Fig. 4: The DnaKJE chaperone system. (A) Three dimensional structure of the E. coli DnaK. (aa 1

to 605) truncated structure of DnaK bound to ADP (Bertelsen et al, 2009) (PDB 2KHO). The N-

terminal NBD (dark green) and the C-terminal SBD (green) are disjointed by a linker (gray). The Lid

of the SBD is in a closed conformation. (B)DnaKJE chaperone cycle. The ATP bound DnaK has a

low-affinity for substrate. The co-chaperone DnaJ mediates the recruitment of substrate to DnaK and

stimulates its ATPase activity. Hydrolysis of ATP, leading to the ADP bound DnaK, induces

allosteric modifications of DnaK allowing the sequestration of the substrate. Then, the Nucleotide

Exchange Factor GrpE facilitates ADP/ATP exchange opening the lid of DnaK and the substrate

release (Genevaux et al,2007).

Page 25: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

24

b- Functions of DnaK

It is believed that DnaK interacts and assists the co- and/or post-translational folding

of about 15% of the nascent peptides or newly synthesized proteins (Deuerling et al, 1999;

Teter et al, 1999). DnaK also participates in the refolding of misfolded and aggregated

proteins, translocation through biological membrane and oligomeric complex

assembly/disassembly (Genevaux et al, 2007; Mayer & Bukau, 2005). DnaK acts on protein

aggregates cooperating with other molecular chaperones: ClpB (Hsp100), Hsp31, or the small

heat shock proteins (sHSPs) IbpA IbpB (Genevaux et al, 2007). DnaK is also involved in the

oxidative stress response and controls the heat shock response by stimulating the σ32

degradation (Hoffmann et al, 2004; Straus et al, 1990). The central role of DnaK is revealed

by deletion of its gene. Indeed, a dnaK null mutant in E. coli exhibits multiple phenotypes

including cryosensitivity beyond 20°C, thermosensitivity above 35°C, filamentation, slow

growth at the permissive temperature of 30°C, resistance to bacteriophages λ P1 and P2

infection, loss of motility, sensitivity to nutrient starvation and defective in plasmid

maintenance (Genevaux et al, 2007). DnaK deletion induces a ribosome biogenesis defect

indicating a role for DnaK in ribosome assembly (Al Refaii & Alix, 2009; Maki et al, 2002).

c- Interactors in the cell

Using a screening of 37 different proteins split in more than 4500 peptides and bound

to a cellulose membrane, the recognition motif of DnaK has been defined as an extended five

residue segment composed of hydrophobic amino acids, Leu, Ile, Val Phe and Tyr

preferentially, framed by positively charged residues (Rudiger et al, 1997). These motifs

occur often in proteins, every 50 to 100 amino acids (Rousseau et al, 2006). Such a variety of

cellular functions and high frequency of binding motifs suggest that DnaK interacts with a

large number of newly synthesized proteins. Indeed, a recent analysis of the DnaK

interactome in E. coli revealed that DnaK interacts with more than six hundred proteins under

physiological condition. 80% of these interactors are cytosolic proteins, but DnaK also

interacts with inner membrane proteins (~11%), outer membrane proteins (~3%) and

periplasmic proteins (~3%) (Calloni et al, 2012). The interaction with exported proteins is in

agreement with the role of DnaK in protein export (Randall & Hardy, 2002). Analysis of the

relative enrichment of substrates on DnaK pointed out that 40% of the total mass of DnaK

substrates was highly enriched on DnaK. These enriched proteins were identified to be below

average cellular abundance and with a low solubility property. These proteins are known to be

Page 26: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

25

aggregation-prone (Tartaglia et al, 2010; Tartaglia et al, 2007). Interestingly, essential

proteins are under the low enriched protein group of DnaK interactors. This is probably due to

the fact that folding of these substrates is supported by the chaperonin GroESL.

3- The chaperonin GroESL

a- Structure of the GroESL complex and chaperonin cycle

The chaperonin GroESL is the only known chaperone system essential in E. coli(Fayet

et al, 1989). GroESL is an asymmetric complex composed by two heptameric rings of 57kDa

monomer of GroEL (Xu et al, 1997). A GroEL barrel is subdivided in three different domains:

the ATPase equatorial domain, the intermediate domain responsible of GroEL rearrangement

after substrate binding, and the apical domain presenting hydrophobic residues involved in

substrate binding and forming the interaction site with the GroES cochaperone (Bukau &

Horwich, 1998). GroES is structured in heptamer of 10kDa subunits and forms a dome

closing one ring of GroEL. Rings are termed as follow: the cis side is composed of a GroESL

complex encapsulating a substrate and the trans side is made by a free and open GroEL

cylinder (Fig. 5A) (Hartl & Hayer-Hartl, 2009).

At the beginning of the cycle, 7 ATP molecules bind to the trans-ring of GroEL. Then

the substrate is recruited to this GroEL ring. ATP molecules are known to induce

rearrangements of the apical domain of GroEL mediating the capture and the compaction of

the substrate to facilitate its encapsulation (Clare et al, 2012; Lin et al, 2008). Then a GroES

dome encloses the substrate into the cage and creates the new cis-ring of the complex. This

cis-ring interaction between GroES and the apical domains of GroEL will enhance the

hydrophobicity of the cavity, inducing the release of the substrate into the cage formed by the

GroEL cylinder. Then, rearrangements of the apical and the intermediate domains in the cis-

ring will bury the hydrophobic residues and change the environment of the cage. The volume

of the cage will also be enhanced and will be able to accommodate polypeptides up to 60kDa

(Hartl & Hayer-Hartl, 2009). The new substrate enclosed into GroESL complex now folds

into a secure and isolated space during 10s to 15s, time required for ATP hydrolysis. Then

release of ADP and GroES will free the substrate. The cycle will repeat from the new trans-

ring and the substrate will be free folded or will necessitate other cycles of folding into

GroESL (Fig. 5B). This GroESL dependent folding may occur in a passive “Anfinsen cage”

(Horwich et al, 2009; Motojima et al, 2012), with an active role via negatively charged

residues in the cavity (Chakraborty et al, 2010; Lin et al, 2008; Tang et al, 2008; Tang et al,

Page 27: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

26

2006) or by forced unfolding (Lin et al, 2008). Action of GroESL mediated folding may differ

in relation to the substrate bound.

b- Interactors in the cell

In vivo, GroESL is known to interact with approximately 10% to 15% of the newly

synthesized cytosolic proteins (Houry et al, 1999). 250 GroESL substrates were identified by

mass spectrometry grouped into 3 different classes using their necessity to be helped by

chaperon systems. The Class I proteins have a relatively low propensity to aggregate and are

chaperone independent. Proteins of Class II need DnaKJE or GroESL to fold properly. Class

III proteins are obligate GroESL substrates These proteins range between 20kDa to 50kDa

and are enriched in (αβ)8 TIM barrel (Kerner et al, 2005). 85 of GroESL substrates are strictly

GroES

GroEL

cis-ring

GroEL

trans-ring

A

B

ADP

7 ATP

+

ATP

GroEL

ATP

GroES

ADP

7 ADP

7 Pi (10-15s)

ADP

7 ATP+

+

7 ADP

Fig. 5: The GroESL chaperonin system. (A) Three dimensional architecture of the GroESL-

7ADP complex (Xu et al, 1997) (PDB 1AON). The GroES heptamer (dark orange) encapsulates

the cavity of the cis-ring of GroEL (orange). The cis-ring of GroEL is in an extended

conformation in comparison to the trans-ring (gray). (B) GroESL chaperonin cycle. Details

concerning the cycle are given in the text.

Page 28: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

27

GroESL-dependent for their folding from which 13 are essential proteins. These 85 substrates

occupy 75% to 80% of GroESL capacity in the cell (Kerner et al, 2005).

Recently, Class III proteins were over-expressed and tested for their dependence to

GroESL in GroESL-depleted cells (Fujiwara et al, 2010). They found that 60% of Class III

proteins are in fact GroESL-obligated substrates, the other 40% can use other chaperone

machineries for their folding under over-expressing conditions. These obligate GroESL-

substrates were grouped as Class IV. Note that these proteins possess a positive bias in

alanine and glycine content, making the proteins more aggregation-prone. Moreover, this

group contains six essential proteins for E. coli: DapA, ASD, MetK, FtsE, HemB and KdsA

(Fujiwara et al, 2010).

Interestingly, in the absence of both TF and DnaKJE, GroESL interacts with about 150

additional substrates (Kerner et al, 2005), thus reflecting interplay among these chaperone

systems.

4- Interplay between TF/DnaKJE/GroESL during de novo protein folding

The molecular chaperones TF, DnaKJE and GroESL are known to work as a network

participating together in the folding of nascent chains and newly synthesized proteins (Fig. 6).

Both TF and DnaKJE most likely share more than 300 proteins as common substrates

(Deuerling et al, 2003). Cooperation between these two chaperones had been shown in vitro

using the multi-domain protein firefly luciferase, with TF and DnaKJE acting co-and post-

translationally, respectively (Agashe et al, 2004). Analysis of the effect of TF, DnaKJE and

GroESL on about 800 cytosolic proteins was carried out using a reconstituted cell-free

translation system (Niwa et al, 2012). In this work, the authors showed that either DnaKJE or

GroESL efficiently increase the solubility of more than 66% of the proteins tested. In this

case, TF only had a minor effect on prevention of aggregation of these substrates. These

results strongly support previous in vitro translation experiment (Agashe et al, 2004) (Fig. 6).

To go further inside the chaperone network, Calloni et al (2012) recently characterized

interactors of DnaK following TF deletion or GroESL depletion. In the absence of TF, DnaK

was found to interact with a higher number of substrates: 998, including 95% of the 674

DnaK interactors already characterized in the presence of TF. This extended substrate

interaction of DnaK in the absence of TF correlates with the overlapping of function already

observed between these two molecular chaperones (Deuerling et al, 1999; Teter et al, 1999),

Page 29: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

28

i.e.in the absence of TF, DnaK interacts with 2 to 3 times more nascent chains than in the

presence of TF (Teter et al, 1999). Note that in this case, DnaK could also interact with

shorter nascent polypeptides (<30kDa) (Deuerling et al, 1999; Teter et al, 1999).

As state above, E. coli tolerates the single deletion of either tig or dnaK(Deuerling et

al, 1999; Genevaux et al, 2004; Teter et al, 1999) and upregulation of DnaKJE is observed in

tig null-mutant (Deuerling et al, 2003). Simultaneous deletions of both tig and dnaK genes

present synthetic lethality at temperatures ≥30°C with respect to the strain background used

(Deuerling et al, 1999; Genevaux et al, 2004; Teter et al, 1999; Vorderwulbecke et al, 2004).

This double mutant possessed a narrow temperature range of growth from 20°C to 30°C,

presented a filamentous phenotype and accumulated aggregated proteins (Deuerling et al,

1999; Genevaux et al, 2004). When only 15 and 474 proteins aggregated in ∆tig and ∆dnaK

single mutants respectively, this amount of aggregated proteins reached 1087 in the absence

of both molecular chaperones (Calloni et al, 2012). A high number of ribosomal proteins were

also found into these aggregates reflecting the strong defect in ribosomal biogenesis of the

∆tig ∆dnaK double mutant (Calloni et al, 2012). This is in agreement with the role of both TF

and DnaKJE in ribosome biogenesis presented above.

GroESL is upregulated in the absence of DnaK and TF and correspondingly, GroESL

depletion induces DnaKJE system and modifies DnaK interactors (Calloni et al, 2012).

Indeed, in this case, 92 proteins had an increased interaction with DnaK; including 38

GroESL substrates of which 19 Class III obligated GroESL substrates. In contrast, 54 proteins

had a reduced interaction with DnaK, including 11 Class II GroESL substrates (Calloni et al,

2012). This suggests that DnaK could substitute its substrates with GroESL-obligated ones,

preventing their aggregation or mediating their degradation.

Remarkably, when both DnaK and TF are absent, GroESL interacts with 150

additional interactors (Kerner et al, 2005). Under these conditions, GroESL can substitute its

substrates for DnaKJE and TF ones. This partial switch of substrates is most likely

responsible of the partial aggregation of several Class III GroESL-obligatory substrates

(Kerner et al, 2005). Supporting such interactome data, GroESL over-expression suppresses

the growth defect of the ∆tig ∆dnaK double mutant (Genevaux et al, 2004; Vorderwulbecke et

al, 2004).

These results indicate that TF, DnaKJE and GroESL are the main players of the

intricate network of molecular chaperones (Fig. 6), in which DnaKJE appears to be a central

Page 30: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

29

node, acting co- and/or post-transnationally downstream of TF and upstream of GroESL

(Calloni et al, 2012).

Part III:Interplays between TF, DnaKJE and GroESL and other

chaperones during protein export

TF

Native

~65-80%

GroESL

~10-15 %~10-20 %

DnaKJE

Fig. 6: Interplay between TF, DnaKJE and GroESL during de novo protein

folding: Nascent polypeptides interact with TF in a co-translational way. A

majority of them (65% to 80%) may reach their native structures without further

assistance. A subset of proteins may need the assistance of other molecular

chaperones to get their native conformation. The DnaKJE system interacts with

polypeptides both in a co- and a post-translational way, mediating the folding of

about 10% to 20% of cytosolic proteins. This chaperone system cooperates with

the chaperonin GroESL by substrate exchange. GroES mediates the folding of

approximately 10% to 15% of cytosolic proteins (Hoffmann et al, 2010).

Page 31: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

30

The major TF, DnaKJE and GroESL chaperones participate in the export of proteins

by acting directly on substrates of the Sec and Tat (Twin-arginine translocation) general

secretion pathways (Graubner et al, 2007; Li et al, 2010; Perez-Rodriguez et al, 2007; Phillips

& Silhavy, 1990; Wild et al, 1992; Wild et al, 1996).

1- Sec Translocation system

The Sec translocon, through which substrates will cross the membrane, is composed of

a hetero-oligomeric complex mainly formed by SecYEG located in the inner membrane (Van

den Berg et al, 2004). SecA is a cytosolic protein promoting the translocation of the substrate

through the Sec translocon in an ATP-dependent manner (Schiebel et al, 1991). In E. coli this

system is supported by the molecular chaperone SecB. SecB facilitates export of OMPs and

some periplasmic proteins which have to be kept unfolded to be exported via the Sec-

translocon (Kumamoto & Beckwith, 1985). In agreement with such specific needs, the

tetrameric SecB chaperone possesses an anti-folding activity (Collier et al, 1988; Randall &

Hardy, 2002). It has been shown that TF, DnaKJE and GroESL play a role during protein

export as well (Altman et al, 1991; Calloni et al, 2012; Lecker et al, 1989; Oh et al, 2011;

Phillips & Silhavy, 1990; Qi et al, 2002; Ullers et al, 2007; Wild et al, 1992; Wild et al, 1996)

(Fig. 7). In addition, functional overlaps and complementarities had been observed between

SecB and theses chaperones during protein export. Indeed, both DnaKJE and GroESL

facilitate the export of several SecB-dependent substrates and their over-expression efficiently

rescue the cold-sensitive phenotype of a secB null mutant (Altman et al, 1991; Phillips &

Silhavy, 1990; Qi et al, 2002; Ullers et al, 2007; Wild et al, 1992; Wild et al, 1996).

Furthermore, the export of several OMPs following SecA depletion is abolished when DnaK

functions are altered (Qi et al, 2002), suggesting that DnaK is able to maintain Sec-dependent

OMPs in a translocation-competent state (Qi et al, 2002). In support of such overlaps, both

secB and dnaK mutations are synthetic lethal and a direct interaction between SecB and DnaK

has been recently identified in vivo(Calloni et al, 2012). Remarkably, SecB over-expression

also suppresses the growth defect and the severe protein aggregation observed in the combine

absence of both TF and DnaK (Ullers et al, 2004). This suppression was independent of SecB

export function as judged by the used of the variant E77K impaired in SecA interaction

(Fekkes et al, 1998; Kimsey et al, 1995).This indicates that in addition to its role in protein

export, SecB can perform generic chaperone functions and facilitate cytosolic protein folding

in the absence of TF and DnaK. Finally, such interplays are further supported by the fact that

Page 32: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

31

SecB endogenous levels are significantly increased upon depletion of DnaKJE and GroESL,

and reciprocally (Muller, 1996).

Although TF binds some OMPs and most likely facilitates their translocation (see Part

II), it has been shown that TF can also antagonize the action of both SecB and DnaKJE during

protein export, as judged by its ability to severely delay export of some Sec-dependent

proteinswhen SecB or DnaKJE are absent (Lee & Bernstein, 2002; Ullers et al, 2007). A

model presenting the different chaperone pathways involved in the Sec-dependent protein

secretion is show in Fig. 7.

2- Twin Arginin translocation system

The second general secretion pathway in E. coli is the Tat system. The translocon is

composed by three membrane proteins of which encoding genes are organized in an operon:

genes encoding for TatA, TatB and TatC; another one, isolated, encodes for TatE and is a

functional TatA duplication (Sargent et al, 1998). This translocon translocates proteins having

the consensus N-terminal motif (S/T)-R-R-X-F-L-K previously folded into the cytoplasm

(Berks, 1996). Majority of Tat substrates have their own specific chaperones called REMPs

(Redox Enzyme Maturation Proteins) like DmsD, NarJ, NarD or TorD (Turner et al, 2004).

They interact specifically with the twin-arginine leader peptide protecting the substrate from

degradation, maintaining it in a translocation competent state and targeting it to the Tat

translocon (Sargent, 2007; Turner et al, 2004). REMPs are not acting alone and participate in

a chaperone cascade also orchestrated by TF, DnaKJE and GroESL (Perez-Rodriguez et al,

2007). Interactions between DmsD and these molecular chaperones were pointed out by

several in vitro experiments (Li et al, 2010). TF, DnaKJE and GroESL were shown to interact

directly with Tat substrates and to efficiently participate in their translocation (Fig. 7). TF was

shown to interact with signal peptides of TorA and SufI until late translation but did not

participate actively in their translocation (Jong et al, 2004). DnaK is essential for the

translocation of over-expressed CueO, and efficiently binds to the leader peptide of DmsA

(Graubner et al, 2007; Oresnik et al, 2001). Moreover, several Tat substrates appear to be in

an unstable conformation in the absence of DnaK (Perez-Rodriguez et al, 2007). These data

suggest that DnaK may work in cooperation with REMPs to stabilize and protect substrates

from degradation. The GroESL interactome revealed the presence of the Tat-dependent

amidase AmiA as a GroESL substrate, which required GroESL for its folding (Kerner et al,

2005; Rodrigue et al, 1996).

Page 33: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

32

SecB

TatABC

Folded

REMP

+

Inner membrane

+

SecA

SecYEG

Cytoplasm

Periplasm

DnaKJEGroESL

TF

Fig. 7: Chaperone networks involved in protein export. Molecular chaperones play different roles in both

systems. In Sec translocation TF, DnaKJE, GroESL, and SecB generally favor secretion by keeping substrates

in an unfolded competent state for translocation. The N-terminal Signal sequence of the substrate is represented

by a green rectangle. In Tat system, the three major chaperones work with REMPs to fold the substrate and

target it to the Tat translocon. The N-terminal Signal sequence of the substrate is represented by a cyan

rectangle.

Page 34: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

33

Part IV: Chaperone-mediated response to protein misfolding and

aggregation

The TF, DnaKJE and GroESL chaperone network is not sufficient to rescue protein

aggregation during heat-stress or oxidative protein unfolding. Other heat shock proteins

(Hsps) are induced in these conditions to facilitate the action of DnaKJE and most likely

GroESL. These chaperones include the Clp superfamily proteins (Hsp100), the small Hsps

(sHsps) IbpA and IbpB, HtpG (the Hsp90 homolog) and the redox-regulated chaperone Hsp33

(Hartl, 1996; Hoffmann et al, 2004; Squires & Squires, 1992).

1- Molecular chaperone networks and protein disaggregation

a- Presentation of other chaperones involved in protein disaggregation

Members of the Clp family include protein complexes functioning as disaggregases

and/or proteolytic machines with AAA+ ATPases activities (ATPases Associated with a

variety of cellular Activities). They can be gathered into two groups depending on the number

of Nucleotide Binding Domains (NBD) they possess. Class I AAA+ proteins comprising

ClpA, ClpB, ClpC and ClpE monomers have two different NBDs separated by a coiled coil

middle domain; Class II proteins with ClpX and ClpY monomers have only one NBD

(Schirmer et al, 1996). Some AAA+ proteins possess a protease domain like FtsH and Lon.

ClpB (Hsp100) possesses a disaggregase activity forming a barrel-shaped hexamer with an

axial channel when it is bound to ATP (Akoev et al, 2004). This channel is surrounded by the

two NBDs of ClpB (NBD1 and NBD2), the middle domain (M domain) of the monomer

being outside of the cylinder (del Castillo et al, 2011; Lee et al, 2003). The N-terminal domain

of each monomer forms a crown involved in the interaction with aggregated proteins (Barnett

et al, 2005). Then ATP hydrolysis will mediate disaggregation by ClpB via translocation of

the substrate through the channel (Weibezahn et al, 2004). The M domain, which is structured

as a coiled coil with two wings, is essential for the disaggregase activity of ClpB (Kedzierska

et al, 2003; Lee et al, 2003; Mogk et al, 2003b). It is involved in the stabilization of the

hexameric structure of ClpB (del Castillo et al, 2011). Seyffer and co-workers (2012) showed

that DnaK could bind the M domain and induce allosteric modifications in ClpB, which,

together with substrate interaction, stimulate ClpB ATPase activity (Seyffer et al, 2012). The

E. coliclpB gene possesses an internal translation initiation site expressing two isoforms of

ClpB in vivo: the full length 95kDa ClpB and a truncated isoform of 80kDa without the N-

terminal domain (Squires et al, 1991). It has been shown that these two isoforms cooperate to

Page 35: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

34

favor solubilization of aggregated proteins and that each isoform alone is less effective in this

task (Guenther et al, 2012; Zhang et al, 2012). The N-terminal domain is involved in the

interaction with aggregates; this combination of isoforms could give more flexibility at this

domain and then facilitate the interaction with substrates (Nagy et al, 2010). The deletion of

clpB in E. coli is known to present a growth defect at 44°C and is also more sensitive to heat

shock at 50°C than the wild type cell (Squires et al, 1991).

The E. coli IbpA and IbpB proteins (Inclusion body protein) are conserved small heat-

shock proteins of 16 kDa that share about 50% amino acid sequence identity and that are

known to associate with thermally aggregated proteins and inclusion bodies (Allen et al,

1992; Laskowska et al, 1996). These two sHsps are known to form molecular oligomeric

structures (Kitagawa et al, 2002; Shearstone & Baneyx, 1999) and cooperate to disaggregate

aggregated polypeptides. The ibpAB operon can be deleted with no effect on cell during

normal growth conditions but causes a decreased viability during prolonged growth at 50°C

(Kuczynska-Wisnik et al, 2002) and as a sensitivity phenotype to copper-induced stress under

aerobic conditions (Matuszewska et al, 2008).

A third chaperone known to participate in disaggregation is the E. coli Hsp90

chaperone member named HtpG. It is an ATP-dependent chaperone functioning as a

homodimer. HtpG monomer is made up of three domains: an N-terminal domain with ATPase

activity, a middle domain and a C-terminal domain involved in Hsp90 dimerization

(Krukenberg et al, 2011; Mayer, 2010; Pearl & Prodromou, 2006; Wandinger et al, 2008).

HtpG is essential for the activity of the clustered regularly interspaced short palindromic

repeats (CRISPR) system(Yosef et al, 2011) involved in the detection and the degradation of

exogenous DNA (Barrangou et al, 2007; Brouns et al, 2008; Marraffini & Sontheimer, 2008)

and RNA (Hale et al, 2009) in prokaryotes. As for clpB, htpG deletion only shows a growth

defect above 44°C (Bardwell & Craig, 1988).

b- Chaperone networks involved in protein disaggregation

In vitro experiments showed that efficient interaction of ClpB with aggregates was

DnaKJE-dependentand thatClpB and DnaKJE systems are strong partners for protein

disaggregation, acting as a bi-chaperone system by which the substrate is transferred from

DnaKJE to ClpB (Acebron et al, 2009; Goloubinoff et al, 1999; Motohashi et al, 1999;

Zolkiewski, 1999). This substrate transfer is mediated by a direct interaction between DnaK

and the M-domain of ClpB (Seyffer et al, 2012). In addition it has been shown that DnaJ

Page 36: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

35

efficiently bindsprotein aggregates independently of DnaK and recruits DnaK to the

aggregates with or without ClpB (Acebron et al, 2008; Winkler et al, 2012). In contrast, ClpB

cannot efficiently bind aggregates in a dnaKmutant (Spence et al, 1990), indicating that the

DnaKJE system first interacts with aggregates and presents the substrates to ClpB. Deletion of

clpB in a dnaK mutant straininduces a strong deleterious effect on E. coli growth when

compared to their respective single mutant controls. Similar results were obtained in a

groES30 mutant allele showing their importance during heat shock (Thomas & Baneyx,

1998). An in vivoFRET (Fluorescence resonance energy transfer) experiment confirmed the

chaperone interaction on aggregates. Indeed, they showed that the DnaJ interaction with

aggregates was followed by recruitment of DnaK. Then DnaK transferred substrates to

ClpB(Kumar & Sourjik, 2012).

The sHsps proteins are strongly recruited to aggregates: 50% of IbpA and IbpB

proteins were bound to aggregates after heat shock at 45°C. This percentage could even reach

80% in ΔclpB and ΔdnaK mutant cells (Mogk et al, 1999), thus revealing the importance of

the chaperone network in the dissolution of aggregates and the reactivation of proteins. How

do IbpA and IbpB favor the dissolution of aggregates mediated by the bi-chaperone

DnaKJE/ClpB? Ratajczak and co-workers (2008) showed that IbpA alone could bind heat-

induced aggregates and reduce their size. However, it could not accelerate the disaggregase

effect of DnaKJE and ClpB. In this case, solitary IbpA even inhibited the downstream action

of the DnaKJE/ClpB bi-chaperone on aggregates, suggesting that it may block the

translocation through the central pore of ClpB. However, IbpB binds to the aggregates only

after IbpA and both IbpA and IbpB favor the action of DnaKJE/ClpB (Kuczynska-Wisnik et

al, 2002). IbpB can thus modify the interaction between IbpA and the aggregates and allow

the action of the bi-chaperone DnaKJE/ClpB (Ratajczak et al, 2009). This cooperation

between the sHsps, ClpB and DnaKJE was additionally tested in vivo using combination of

mutations in these molecular chaperones. In this case the solubilization of protein aggregates

was delayed but not abolished in a ΔibpAB strain when compared with wild type, indicating

IbpA and IbpB are not essential to the solubilization of aggregates but may facilitate the

action of ClpB and DnaKJE (Mogk et al, 2003a). Yet, as observed for clpB mutations (see

above), the combined deletion of ibpABand dnaK exhibitsa strong growth defect when

compared to the respective single mutants(Thomas & Baneyx, 1998). Furthermore, Kumar

and Sourjik (2012) showed that, during severe denaturation conditions, when the DnaKJE

chaperone system is strongly titrated to aggregated or unfolded proteins, both IbpA and IbpB

Page 37: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

36

localize with aggregated proteins. ClpB is then recruited to aggregates but DnaKJE is needed

for efficient disaggregation (Fig. 8).

As for ClpB and IbpA/B, HtpG cannot reactivate denatured luciferase in vitro alone.

But its presence significantly stimulates refolding by DnaKJE, up to 1,6-fold when compared

to DnaKJE alone (Genest et al, 2011). This mode of action takes place in a sequential manner,

by first binding of DnaK and the subsequent recruitment of HtpG to the substrate (Genest et

al, 2011). Although the effect of HtpG on aggregates in not well defined yet,the deletion of

htpGeither in a dnaK or a groES30 mutant further affects bacterial growth, thusfurther

arguing for a role for HtpG in the disaggregation pathway (Thomas & Baneyx, 1998).

Together these data reveal the importance of the cooperation between ClpB, IbpA

IbpB and HtpG with the DnaKJE chaperone systems in protein disaggregation (Thomas &

Baneyx, 1998).

IbpAB

oligomersAggregates

ClpB

Native

Proteases

+

DnaKJEGroESL

Unfolded protein

DnaKJ

Fig. 8: Mainmolecular chaperones functions in disaggregation. IbpA (in green) and IbpB (in violet)

oligomers break aggregates into smaller structures. DnaKJ starts to free proteins and cooperate with

the disaggregase ClpB to solubilize them. Then, disaggregated proteins may reach their native

structure with or without the action of DnaKJE and GroESL. If their folding is not possible, because of

a titration of molecular chaperones or an irreversible modification, they will be degraded by proteases.

Page 38: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

37

2- Hsp33 as a member of the chaperone network

As stated above, the simultaneous deletion of both dnaK and tig leads to a severe

growth defect and protein aggregation (Deuerling et al, 1999; Genevaux et al, 2004; Teter et

al, 1999; Vorderwulbecke et al, 2004). This strain gives an advantage to study chaperone

networks and to further elucidate interactions between molecular chaperones in vivo. Both

SecB and GroESL have been previously identified as suppressors of these phenotypes

(Genevaux et al, 2004; Ullers et al, 2004; Vorderwulbecke et al, 2004). In this thesis work,

another protein suppressing these sensitive phenotypes has been identified: the molecular

chaperone Hsp33.

a- Hsp33 is involved in the oxidative stress response

Oxidative stress is concomitant with life in aerobic conditions, favoring production of

reactive oxygen species O2- or H2O2. This stress can be induced under culture conditions by

adding H2O2 or HOCl in the medium or after heat shock (Imlay, 2003; Storz & Imlay, 1999;

Winter et al, 2008). Oxidative stress is responsible of the oxidation of all macromolecules in

the cell: DNA, lipids and proteins (Imlay, 2003). This stress even paralyzes the chaperone

network functions inactivating the DnaK molecular chaperone system (Winter et al, 2005).

Proteins oxidize mostly on sulfur group of cysteines and methionines, increasing their

susceptibility to aggregation and degradation (Storz & Imlay, 1999). Some proteins involved

in the oxidative stress response become active through disulfide bound formation (Aslund &

Beckwith, 1999). This is the case of the transcriptional regulators OxyR and SoxR inducing

the oxidative stress response (Ding & Demple, 1998; Zheng et al, 1998). OxyR upregulates 28

genes involved in the protection against oxidative stress such as glutathione oxidoreductase,

glutaredoxin I, Catalase/hydroperoxidase I, Thioredoxin reductase and Thioredoxin 2 (Chiang

& Schellhorn, 2012). SoxR oxidative stress response is involved in superoxide radical

response. SoxR activated by an oxidative stress induces the expression of the transcriptional

regulator SoxS known to regulate the expression of more than 100 genes including the

manganese-superoxide dismutase sodA, the endonuclease IV involved in DNA repair and

yggX protecting iron-sulfur proteins against oxidative stress (Blanchard et al, 2007). A

molecular chaperone was also found to be activated by oxidative stress and participate to this

stress response, the heat shock induced Hsp33 (Jakob et al, 1999).

Page 39: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

38

b- Structure of Hsp33

Hsp33 is a very well conserved protein possessing homologues in many prokaryotes

species and also in some eukaryotic unicellular organisms like Trypanosomatidae(Jakob et al,

1999). Synthesis and activity of this protein is regulated by a two level mechanism: the heat

shock response for the transcriptional regulation and an oxidative stress response for its

activation via disulfide bound formation. This modification is reversible (Jakob et al, 1999).

Hsp33 is a 32,9kDa protein structured in three different domains (Fig. 9). The N-

terminal domain of Hsp33 (aa 1-178) is folded as a core domain, very compact, with two anti-

parallel β-sheets of five and four β-strands (Graf & Jakob, 2002). This domain presents some

structural homology with the TF ribosome binding domain except the absence of the loop

region responsible for the ribosome binding (Kristensen & Gajhede, 2003). The linker region

(aa 179-232) is folded in the reduced form of Hsp33 and interacts with the N-terminal domain

(Janda et al, 2004; Vijayalakshmi et al, 2001). The C-terminal domain (aa 232-294) contains

four cysteines absolutely conserved in all Hsp33 homologues (organized as follow: C232

-X-

C234

-(27-32)-C265

-X-Y-C268

) stabilizing a zinc ion in a tetrahedral conformation. This domain

is named the zinc center (Graf et al, 2004; Jakob et al, 2000). This domain is known to be the

oxidative sensor domain of Hsp33 (Jakob et al, 1999).

Page 40: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

39

c- Activation cycle of Hsp33

Hsp33 is an ATP-independent molecular chaperone acting as a holdase to prevent

aggregation of oxidized and denatured proteins rather than efficiently participate in their

folding (Jakob et al, 1999). The redox potential of a cell under normal growth conditions is

ranged between -250mV and -280mV keeping Hsp33 inactive (redox potential of -170mV ±

10mV) (Jakob et al, 1999). When an oxidative stress occurs, the cellular redox potential is

about to reach >-150mV which allows the activation of Hsp33 (Gilbert, 1990).

Zn2+

178

N

232 294

C

N-terminal

domain

Linker

zinc center

A

B

Fig. 9: Hsp33 structure. (A)

Three dimensional structure of

reduced Hsp33 ribbon diagram

with a Zn2+

ion bound to the zinc

center (PDB 1VZY). (B) Domain

arrangement of Hsp33 showing

the 4 strictly conserved cysteins

in the zinc center. The N-

terminal is shown in blue, the

linker region in green and the

Zinc center in orange (Kumsta &

Jakob, 2009).

Page 41: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

40

The activation of Hsp33 as a molecular chaperone is a two-step process. At the

beginning, reduced Hsp33 is in a monomeric state and the zinc center is folded. When an

oxidative stress occurs, disulfide bound formation is formed between Cys265

and Cys268

releasing the zinc ion and unfolding the zinc center domain (Ilbert et al, 2007; Won et al,

2004). Hsp33 will stay in this oxidized non-active conformation until a denaturing stress

happens. Heat stress favors the unfolding of the linker region and facilitates the disulfide

bound formation between Cys232

and Cys234

unfolding the linker region (Leichert et al, 2008).

Then, activated Hsp33 interacts with its substrates, between the linker region and the N-

terminal domain (Reichmann et al, 2012), as a monomer (Barbirz et al, 2000), a dimer

(Vijayalakshmi et al, 2001) or an oligomeric structures (Akhtar et al, 2004) depending the

oxidized Hsp33 concentration and the temperature (Akhtar et al, 2004; Graf & Jakob, 2002).

The dimerization of activated Hsp33 is favored by high temperature (Akhtar et al, 2004;

Graumann et al, 2001). An in vitro experiment revealed that a small population of Hsp33

oligomers (until octamer) may occur with higher temperature and in late time of oxidative

stress (Akhtar et al, 2004). These authors showed that oligomers of Hsp33 were more efficient

than the dimeric conformation for the protection of thermally unfolded citrate synthase.

Presence of this oligomeric structure is not yet proved in vivo. But during an oxidative stress,

Hsp33 is present in high concentration, which could favor apparition of this structure.

The reversible inactivation of Hsp33 necessitates at the same time reducing and

refolding conditions (Ilbert et al, 2006). Inactivation of the Hsp33 dimer-substrate complex by

glutaredoxin and thioredoxin doesn’t release the substrate into the cytosol. Release of the

substrate from the reduced-Hsp33 requires a functional DnaKJE system (Hoffmann et al,

2004). This substrate exchange mediates the folding of denatured proteins when stress

conditions are over and when sufficient amount of DnaKJE system is reactivated (The Hsp33

chaperone cycle as a dimer is presented in Fig. 10). Oxidative stress is known to drop the ATP

concentration in the cell (Osorio et al, 2003). This ATP depletion combined to oxidative and

heat stresses inactivate DnaK by unfolding of its NBD. This inactivation is reversible when

reducing conditions are restored (Winter et al, 2005). Hsp33 protect unfolded proteins against

aggregation until DnaK’s activity is restored at reduced conditions.

Page 42: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

41

Oxidized Hsp33 molecular chaperone activity has been tested in vitro on several

substrates. It can prevent from aggregation denatured citrate synthase, luciferase or the E. coli

protein FtsJ and the eye-lens δ-crystallin (Jakob et al, 1999; Raman et al, 2001). In vivo,

Hsp33 had been found to protect about 80% of aggregated-prone proteins aggressed by

oxidative stress, including metabolic pathways enzymes, fatty acid biosynthesis and proteins

involved in transcription and cell division. Membrane proteins were not protected by Hsp33

(Winter et al, 2005). More recently, binding specificity of Hsp33 was studied by Reichmann

and coworkers (2012). They found that Hsp33 does not seem to have binding sequence

specificity but prefers interact with secondary structured regions, mostly alpha helices. This

secondary structure interaction preference of Hsp33 may avoid the unproductive interaction

between activated Hsp33 and its unfolded C-terminal domain. Although, there is some

overlapping of substrates between DnaKJE and Hsp33; but DnaKJE only interacts with

unfolded proteins whereas Hsp33 binds structured regions (Jakob et al, 1999; Reichmann et

al, 2012). The reduction of Hsp33 induces conformational changes in the molecular

Heat

stress

Hsp33 Dimerization and

interaction with the substrate

Zn2+SS

SS

Reduced

Hsp33

Reducing and physiological

temperature conditionsSS

SS

SS

SS

H2O2 or

HOCL Zn2+

Oxidative

stress

DnaKJE

Native

Activated

Hsp33

Zn2+

Fig. 10: Hsp33 cycle. An oxidative stress is sensed by the Zinc center of Hsp33 via disulfide

bound formation between Cys 265 and 268 removing the Zn2+

ion from the zinc center. Then,

Hsp33 is fully activated by an heat shock inducing the disulfide bound formation of Cys 232 and

234 which unfolds the linker region and the zinc center. This activation mediates Hsp33

dimerization and interaction with unfolded substrates with secondary structures. When reducing

and physiological temperature conditions are recovered, all cysteines of Hsp33 are reduced;

binding of the Zn2+

ion refolds the linker region and the zinc center. This conformational

modification of Hsp33 dimer mediates the unfolding of its secondary structured substrate

allowing its transfer to the DnaKJE chaperone system. Then Hsp33 returns to its monomeric

conformation. Colors are identical to Fig. 9.

Page 43: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

42

chaperone refolding the zinc center. These conformational modifications trigger unfolding of

its substrate, loss of stability of the Hsp33-substrate complex and finally allow interaction

with DnaK (Reichmann et al, 2012) or with proteases as Lon (Bruel et al, 2012).

d- Hsp33 deletion and functions in the cell

The Hsp33 encoding gene hslO is located in an operon (Graf & Jakob, 2002). Two

other genes are co-translated, hslR and hslP. hslR encodes for the Hsp15 protein, known to

bind and recycle the complex formed by the 50S ribosomal subunit, the nascent chain and the

tRNA after aborted protein synthesis and hslP encodes for a putative phosphatase (Korber et

al, 2000). hslO can be deleted in E. coli and confers an oxidative sensitivity; this effect is

exacerbated in a cell which is always under oxidative stress after deletion of trxB encoding the

thioredoxin. This in vivo result pin points a key role of Hsp33 during oxidative stress (Winter

et al, 2005). Interestingly, hslO deletion in Vibrio cholerae conduces to thermosensitivity

phenotype and a higher sensitivity to oxidative stress. An in vivo experiment showed that

these phenotypes were suppressed by the E. coli EF-Tu over-expression (Wholey & Jakob,

2012). EF-Tu is known to be subject to thiol modification during oxidative stress and partial

depletion of EF-Tu increases the bleach sensitivity of E. coli(Leichert et al, 2008). In addition,

Hsp33 deletion favored the degradation of the V. Cholerae EF-Tu because of a higher

sensitivity to oxidative stress than E. coli EF-Tu (Wholey & Jakob, 2012).

In this thesis work, we showed that Hsp33 efficiently suppressed the bacterial growth

and protein folding defects observed in the absence of both TF and DnaK, thus revealing a

role for Hsp33 as a major participant of the E. coli chaperone network. However, in contrast

with previous results concerning GroESL and SecB, we now showed that Hsp33 acts

indirectly, by specifically interacting with the essential elongation factor Tu (EF-Tu) and

targeting it for degradation by the protease Lon.

Page 44: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

43

MATERIALS

AND

METHODS

Page 45: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

MATERIALS AND METHODS

1- Strains and plasmids

a- Bacterial Strains, phages, and culture conditions

Genetic experiments were carried out in the E. coli MC4100 (Casadaban, 1976) or W3110

genetic background strains (Bachmann, 1972). The MC4100 mutant derivatives Δtig::CmR,

ΔdnaKdnaJ::KanR; Δtig::Cm

R ΔdnaKdnaJ::Kan

R, dnaJ::Tn10-42, Δtig dnaJ::Tn10-42 (Genevaux

et al, 2004), Δtig::CmS (Ullers et al, 2007), Δlon::Kan

R(Sakr et al, 2010), ΔclpP::Cm

R (Maurizi et

al, 1990), ΔclpQ::TetR(Yamaguchi et al, 2003) and W3110 Δtig ΔdnaKdnaJ::Kan

R(Genevaux et al,

2004)have been previously described. The ΔhslO::KanR allele was obtained from strain JWK5692

(Keio collection). All mutations described in this study were moved to the appropriate genetic

background by bacteriophage P1-mediated transduction. Bacteria were routinely grown in LB

medium supplemented when necessary with either chloramphenicol (10 μg/ml), kanamycin (50

μg/ml), ampicillin (100 μg/ml), or tetracycline (15 μg/ml).

The construction of the dnaK-protease and dnaK hslO double mutants was performed as

follows. The KanR cassettes from MC4100 ΔhslO::Kan

R and Δlon::Kan

R were first removed using

plasmid pCP20 as described (Datsenko & Wanner, 2000). The ΔdnaKdnaJ::KanR

mutant allele was

then introduced into the hslO and the various protease mutants, thus leading to the construction of

strains MC4100 ΔhslO ΔdnaKdnaJ::KanR, Δlon ΔdnaKdnaJ::Kan

R, ΔclpP::Cm

R

ΔdnaKdnaJ::KanR, and ΔclpQ::Tet

R ΔdnaKdnaJ::Kan

R. To construct strain MC4100 ΔhslO

Δtig::CmRdnaJ::Tn10-42, the Δtig::Cm

R mutant allele was first introduced intoMC4100 ΔhslO,

followed by introduction of dnaJ::Tn10-42(TetR). In this case, selection for transductants was

carried out at 30°C.

The mutant strains used for the Lon-dependent suppression experiments were constructed as

follows. The ΔhslO::KanR mutant allele was first introduced into MC4100 Δlon. Next, the

ΔdnaK52::CmR mutant allele (Bukau & Walker, 1989) was introduced into both MC4100

ΔhslO::KanR and MC4100 Δlon ΔhslO::Kan

R. MC4100 Δtig::Cm

R Δlon::Kan

RdnaJ::Tn10-42 was

constructed by first moving the Δtig::CmR mutant allele into MC4100 Δlon::Kan

R. Note that the lon

and tig genes are 90% linked by P1 transduction on the E. coli chromosome. The dnaJ::Tn10-42

allele was then introduced into MC4100 Δtig::CmR Δlon::Kan

R and selection was done at 30°C. All

DNA cloning and transformations experiments were carried out in strain DH5α (Invitrogen).

Page 46: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

45

b- Plasmid construction

Plasmids pSE380ΔNcoI, p29SEN (Genevaux et al, 2004) and pBAD22 (Guzman et al,

1995) have been previously described. To construct the high-copy number plasmid pSE-Hsp33

(pSE380 ΔNcoI-Hsp33) and the low-copy number plasmid p29-Hsp33 (p29SEN-Hsp33), the 879

bp hslO gene was PCR-amplified using primers Hsp33-for (5'-

CGGAATTCTCATGATTATGCCGCAACATGACC-3’) and Hsp33-rev (5’-

CGAAGCTTGGATCCTTAATGAACTTGCGGATCTGC-3’) using MG1655 genomic DNA as

template. The PCR fragment was digested with EcoRI and HindIII and cloned into either

pSE380ΔNcoI or p29SEN previously digested with the same enzymes. A similar cloning procedure

was used to construct the pSE-Hsp33(Y12E), pSE-Hsp33(M172S) mutant derivatives, except that

in this case, plasmids pET11a-hslO(Y12E) or pET11a-hslO(M172S) DNA was used as a template

(Cremers et al, 2010). Plasmid pSE-Hsp33-FLAG (pSE380ΔNcoI-Hsp33-flag tagged) containing

Hsp33 with the C-terminal FLAG tag “GSDYKDDDDKSA” was constructed using primers Hsp33-

for and 33FT-rev (5’-

GCAAGCTTGGATCCTTAGGCGCTTTTATCGTCGTCATCTTTGTAGTCGCTGCCATGAAC

TTGCGGATCTGC -3’) and pSE-Hsp33 as DNA template. The PCR fragment was cloned as an

EcoRI-HindIII digested PCR fragment in pSE380ΔNcoI digested with the same enzymes. Plasmid

pSE-Hsp33(Q151E) was constructed by quick change mutagenesis using the appropriate primers.

Plasmid pSE-Hsp33(1-235) was constructed using primers Hsp33-for and Hsp33(1-235)-rev (5’-

CAAGCTTGGATCCttaCGAGCAGGTGCATTTGAACTC -3’) and pSE-Hsp33 as DNA template.

The PCR fragment was cloned as an EcoRI-HindIII digested fragment into pSE380ΔNcoI digested

with the same enzymes.

The p29SEN-derivatives containing either the yrfG hslR hslO or the hslR hslO operon were

constructed using yrfG-For (5’- GAGAATTC CATATGCATATCAACATTGCCTG -3’) or hslR-

For (5’-GAGAATTC CATATGAAAGAGAAACCTGCTGTTG -3’) forward primers, respectively.

In this case, the primer Hsp33-rev was used as reverse primer.

To construct plasmid p29-EF-Tu, the 1185bp long tufA gene, encoding EF-Tu, was PCR-

amplified using primers tufA-for (5’- GAGAATTCATGTCTAAAGAAAAATTTGAAC-3’) and

tufA-rev (5’- GAAAGCTTTTAGCCCAGAACTTTAGCAAC-3’) and MG1655 genomic DNA as

template. The PCR fragment was digested with EcoRI and HindIII and cloned into p29SEN

digested with the same enzymes.

The toxin gene hipA (1323bp) was PCR amplified using primers HipA-For (5’-

GCGAATCCATGCCTAAACTTGTCACTTG-3’) and HipA-Rev (5’-

Page 47: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

46

GCGCATGCTCACTTACTACCGTATTCTC-3’) and MG1655 genomic DNA as template. The

hipA gene was then cloned under the control of an arabinose-inducible promoter as an EcoRI-SphI

fragment into plasmid pBAD22 digested with the same enzymes. The resulting plasmid was named

pBAD-HipA. Note that hipA was cloned under the tightly regulated araBADp promoter to minimize

background expression level of HipA in the absence of inducer, thus avoiding killing by the toxin.

All constructs generated by PCR were sequenced verified.

2- In vivo experiments

a- Bacterial viability assay and genetic experiments

In vivo complementation of the temperature sensitive phenotype of the MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR, MC4100 Δtig dnaJ::Tn10-42, MC4100 Δtig ΔlondnaJ::Tn10-42 and MC4100

Δtig ΔhslOdnaJ::Tn10-42, was performed as follows. Cultures of fresh transformants were first

grown overnight in LB ampicillin glucose 0.4% at the permissive temperature (see Figure legends),

diluted 1/50 into the same medium, further grown to mid-log phase, serially diluted ten-fold and

spotted on LB ampicillin agar plates with or without IPTG (at 5, 50 or 500 µM) and incubated at the

indicated temperatures. Complementation of the W3110 ∆dnaKdnaJ::KanR ∆tig::Cm

R temperature-

sensitive phenotype by HipA was carried out as described above, except that L-arabinose inducer

(0.1, 0.5, 1, or 2%) was used in this case. To monitor Hsp33 toxicity of the MC4100 and its mutant

derivatives, mid-log phase cultures of fresh transformants were grown at the permissive growth

temperature (22°C or 30°C; see Figure legends) in LB ampicillin glucose 0.4%, were serially

diluted ten-fold and spotted on LB ampicillin agar plates with or without IPTG (100 or 500 µM),

and incubated at the indicated temperatures.

The co-transduction frequencies between ΔdnaKdnaJ::KanR and the linked thr::Tet

R alleles

were analyzed using bacteriophage P1-mediated transduction as described previously (Teter et al,

1999), except that MC4100 Δtig and MC4100 Δtig ΔhslO were used as the recipient strains.

b- Isolation of protein aggregates and cell fractionation

Cultures of fresh transformants of MC4100 ∆dnaKdnaJ::KanR ∆tig::Cm

R containing

plasmids pSE380∆NcoI, or pSE-Hsp33 wild type or its mutant derivatives were grown at 22°C,

diluted 1/50 and grown to an OD600 of 0.3 in LBampicillin (100 µg/ml) glucose (0.4%). IPTG

inducer (0.5 mM) was added and cultures were further incubated for 2 h at 22°C. Next, the cultures

were transferred to a 37°C water bath, and shaken (180 rpm) for 1 h and protein aggregates were

isolated as described by Tomoyasu and colleagues (Tomoyasu et al, 2001b). Protein aggregates

Page 48: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

47

were prepared and analyzed by SDS-PAGE using 4–15% Mini-Protean TGX gels (Bio-Rad). For

HipA-mediated suppression of protein aggregates, cultures of fresh transformants of W3110

∆dnaKdnaJ::KanR ∆tig::Cm

S carrying plasmid pBAD22, or pBAD-HipA at 22°C were diluted 1/50

and grown to an OD600 of 0.3 in LBampicillin (100 µg/ml). L-arabinose inducer (0.5%) was then

added and cell cultures were further incubated for 4 h at 22°C. Cultures were then transferred to

37°C in a water bath, shaken (180 rpm) for 1 h and protein aggregates were prepared as described

(Tomoyasu et al, 2001b).

c- In vivo pull-down assay

E. coli MC4100, MC4100 ∆tig::CmR, MC4100 ∆dnaKdnaJ::Kan

R, and MC4100 ∆tig::Cm

R

∆dnaKdnaJ::KanR transformed with pSE-Hsp33FLAG were first selected at 22°C on LB agar plates

supplemented with glucose (0.4%) and ampicillin. Overnight cultures of fresh transformants, grown

at 22°C, were diluted to an OD600 of 0.1 and incubated until mid-log phase at 22°C in LB

supplemented with glucose 0.4% and ampicillin. Expression of Hsp33FLAG and

Hsp33(Y12E)FLAG was induced by the addition of 0.5 mM IPTG for 30 min at 22°C and

transferred to 30°C for 1 h and 30 min. Cells were collected by centrifugation (5000g for 5 min at

4°C) and resuspended in 500 µL of cold PD buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mg/mL

lysozyme, and protease inhibitors from Roche) supplemented with Benzonase (3 units/mL culture

volume; Invitrogen). Crude cell extracts were obtained following sonication and centrifugation

(14000g for 30 min). Pull-down assays were performed with 500 µL aliquots of cell extract and 50

µL of Anti-Flag M2-Agarose resin (Sigma) in TBS buffer (150 mM NaCl, 50 mM Tris pH 7.5).

Suspensions were gently rocked at 4°C for 2 h, and beads were pelleted by centrifuging for 1 min at

5000g. After washing the beads 3 times with 1 mL of TBS buffer, proteins were eluted in TBS

buffer containing 100µg/mL of peptide Flag (DYKDDDDK). Samples were separated by SDS-

PAGE on 4–20% Mini-Protean TGX gels (Bio-Rad) and stained with Coomassie instant blue

(expedeon).

3- In vitro experiments

a- Western blot analysis

Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride

membrane (Hybond-P, GE Healthcare) using a semidry transfer system (Trans-Blot SD, Bio-Rad)

for 30 min at 10 V.Membranes were blocked for 1 h at RT or overnight at 4°C with 3% nonfat

milk-Tris-buffered saline with Tween 20 (TBS-T; 0.5 M Tris, 1.5 M NaCl, pH 7.4, plus 0.05%

Page 49: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

48

Tween 20). Rabbit polyclonal antibody against Hsp33 (1:2000 dilution; a kind gift of Ursula Jakob,

University of Michigan), a mouse monoclonal antibody against EF-Tu (1:10000 dilution; Hycult

Biotech), a rabbit antibody against DnaJ (1:3000 dilution; (Genevaux et al, 2002)) and a rabbit

antibody against TF (1:8000 dilution; (Ullers et al, 2007)) were used as primary antibodies, and

HRP-conjugated rabbit IgG (1:5000; Sigma) or mouse IgG (1:2,000; Sigma) were used as

secondary antibodies. Membranes were developed by chemiluminescence using an ECL Plus

(Prime) immunoblotting detection kit following the manufacturer's protocol (GE Healthcare) with a

luminescence analyser (LAS4000, Fuji).

b- In vitro translation and cross-linking experiments

Strains MC4100 ∆dnaKdnaJ::KanR and ∆tig::Cm

R ∆dnaKdnaJ::Kan

R were first transformed

with pSE-Hsp33FLAG and grown in LB ampicillin medium at 30° and 25°C, respectively, were

used to obtain Hsp33-FLAG enriched translation lysates, as previously described for the SecB

chaperone (Ullers et al, 2004). RpoB150 mRNA was prepared as previously described (Ullers et al,

2004). In vitro translation in the E. coli cell- and membrane-free S-135 extract was carried out using

RpoB150 mRNA as previously described (Ullers et al, 2004; Urbanus et al, 2001). Bifunctional

cross-linking was induced by the addition of 1mM bis(sulfosuccinimidyl) suberate (BS3) for 10 min

at 26°C and quenched at 4°C by adding 1/10 volume of quench buffer (1 M glycine, 100 mM

NaHCO3, pH 8.5). Ribosome-nascent chain complexes were collected as described (Ullers et al,

2003) and analyzed either directly following SDS-PAGE (15%) separation or after

immunoprecipitation (Luirink et al, 1992) with a mouse antibody anti-FLAG (Sigma Aldrich).

c- Pulse-chase and immunoprecipitation analyses

Overnight cultures of MC4100 ∆hslO ∆dnaKdnaJ::KanR transformed with either pSE380 or

pSE-Hsp33 were diluted 1/50 in LB ampicillin, in the presence of 100 µCi/ml 35

S-methionine and

grown until the OD600 reached 0.4. Unlabeled methionine was then added at a final concentration of

15 mM and IPTG at 500 µM to induce expression of Hsp33. Samples were taken at time 0, 60, 120

min and kept overnight on ice cold TCA (10%), and analyzed by immunoprecipitation with anti-

EF-Tu antibodies as described (Jong et al, 2004). The gels were scanned using Fuji FLA-3000

phosphorimaging technology.

Page 50: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

49

RESULTS

Page 51: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

50

RESULTS

1- Hsp33 overproduction supports bacterial growth and prevents protein aggregation in

the absence of TF and DnaK

The ATP-independent and redox-regulated molecular chaperone Hsp33 (heat-shock protein

of 33 kDa), which is specifically activated by oxidative protein unfolding, appears to be a key

component of the network of heat-induced stress chaperones and proteases in E. coli(Chuang &

Blattner, 1993; Jakob et al, 1999). Specifically, in the presence of stressors such as hypochlorite or

hydrogen peroxide combined with high temperature, sensitive pairs of cysteine residues in Hsp33

are sequentially oxidized, leading to Hsp33 dimerization and activation of its chaperone function.

The resulting Hsp33 dimers bind unfolded substrate proteins and protect them from aggregation

(Jakob et al, 1999). Upon return to reducing conditions, Hsp33-bound substrates are most likely

delivered to DnaK for their subsequent refolding (Hoffmann et al, 2004).

To highlight a possible role of Hsp33 generic chaperone function in the absence of either

DnaK or oxidative stress, we asked whether Hsp33, as part of the chaperone network in E. coli,

could also restore bacterial growth of the severely compromised Δtig ΔdnaKdnaJ chaperone mutant

at higher temperatures (Genevaux et al, 2004). This strain lacks the TF/DnaK folding pathway and

thus represents a valuable tool for the identification of in vivo chaperone function. The bacterial

colony forming experiments presented in Fig. 1A show that indeed, over-expression of Hsp33

(encoded by the hslO gene, the last member of the yrfG-hslR-hslO operon) enables the MC4100

Δtig ΔdnaKdnaJ chaperone mutant to survive at the otherwise non-permissive temperatures of

33°C. In addition, we noticed that Hsp33 over-expression interferes somewhat with overall bacterial

growth, as judged by smaller colony size formation at the permissive temperatures of growth (Fig.

1A; see below). A western blot analysis showing Hsp33 expression levels in the presence of IPTG

inducer at the permissive temperature of 22°C is provided in Fig. 2. Further analysis of the extent of

assistance by Hsp33 in the absence of TF and DnaK showed that suppression by Hsp33 is not strain

background specific and is not further improved by the expression of the full yrfG-hslR-hslO operon

(Fig. 2).

Page 52: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

51

As stated above, de novo protein folding is severely impaired in the absence of both TF and

DnaK chaperones. As a consequence, proteins massively accumulate as cytoplasmic aggregates in

the Δtig ΔdnaKdnaJ mutant strain. We took advantage of this observation to ask whether Hsp33

over-expression can also prevent the in vivo protein aggregation that occurs in the absence of TF

50 µM IPTG

37ºC

B

250 kDa -

130 -

100 -

70 -

55 -

35 -

25 -

17 -

11 -

WCE Aggregates

22ºC

33ºC

no IPTG

A

dilution

10-6

10-4

10-2

Hsp33

Fig. 1: Hsp33 over-expression rescues bacterial growth and prevents protein aggregation

in the absence of DnaK and TF. (A) Fresh transformants of strain MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

containing the plasmid pSE380∆NcoI parental vector, or pSE-Hsp33, or

pSE-Hsp33(Y12E), or pSE-Hsp33(M172S) or pSE-Hsp33(Q151E) were grown at 22°C,

serially diluted ten-fold and spotted on LB ampicillin agar plates with or without IPTG

inducer. Plates were incubated for 1 day at 33°C or 2 days at 22°C. (B) Protein aggregates and

their corresponding whole cell extracts (WCE) were isolated from strain MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

expressing the pSE-based Hsp33 derivatives, grown at 22°C and transferred

for 1 h at 37°C in the presence of IPTG to induce Hsp33 over-expression. The position of the

protein bands corresponding to Hsp33 or its mutant derivatives is indicated.

Page 53: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

52

and DnaK, as previously shown for the overproduction of SecB or GroESL (Ullers et al, 2007).

Hsp33 was over-expressed in the MC4100 Δtig ΔdnaKdnaJ strain and aggregated proteins were

detected following 1 h incubation at the non-permissive temperature of 37°C, as described

(Tomoyasu et al, 2001a). As anticipated from the bacterial viability results, Hsp33 over-expression

efficiently prevented the accumulation of protein aggregates at 37°C (Fig. 1B).

We next asked whether mutations in the hslO gene, which were previously shown to

differentially affect activation of its Hsp33 chaperone function in vitro, could also rescue the defects

of the Δtig ΔdnaKdnaJ mutant in the absence of oxidizing conditions. Specifically, we chose the

following hslO mutations: Hsp33(Y12E), which is constitutively active in vitro but mainly

insoluble in vivo, Hsp33(M172S), activated by high temperature in the absence of oxidizing

conditions (Cremers et al, 2010), Hsp33(Q151E), constitutively monomeric and redox-regulated

with preference for slowly unfolding protein substrates (Chi et al, 2011), and Hsp33(1-235),

33ºC

22ºC

CA B

33ºC

22ºC- Hsp33

Fig. 2: Hsp33 promotes bacterial growth in the absence of DnaK and TF. (A) Western blot

analysis showing steady state expression levels of chromosomally- (vector) and plasmid- (pSE-

Hsp33) encoded Hsp33 in strain MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

after 6 h induction at

22°C in the presence of 50 µM IPTG and revelation using a rabbit polyclonal anti-Hsp33

antibody.(B) In vivo complementation of W3110 ∆tig::CmR

∆dnaKdnaJ::KanR

temperature-

sensitive phenotype by p29-Hsp33 in the presence of 500 µM IPTG, using the procedure

described in Fig. 1. (C) In vivo complementation in the presence of 500 µM IPTG, of MC4100

∆tig::CmR

∆dnaKdnaJ::KanR

temperature-sensitive phenotype by p29-Hsp33 (hslO) alone, with

Hsp15 (hslR-hslO) or in the context of the whole yrfG-hslR-hslO operon.

Page 54: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

53

constitutively active as a dimer in vitro(Kim et al, 2001). The corresponding mutant genes were

cloned in a multicopy plasmid under the control of an IPTG-inducible promoter and tested for their

ability to suppress the growth defects exhibited by the MC4100 Δtig ΔdnaKdnaJ mutant strain. We

found that, in sharp contrast to the Hsp33 wild-type, over-expression of either the Hsp33(Y12E), or

Hsp33(M172S), or Hsp33(1-235) mutant protein did not rescue bacterial growth at 33°C (Fig. 1A;

Fig. 3). These results suggest that the integrity of the redox switch mechanism is critical for the

Hsp33-mediated suppression of the Δtig ΔdnaKdnaJ phenotype. Consistently, neither the

Hsp33(Y12E) nor the Hsp33(M172S) mutant was capable of preventing protein aggregation at

37°C in vivo (Fig. 1B). Instead, these two mutant Hsp33 proteins co-purified with the protein

aggregates. Although it is known that Hsp33(Y12E) is mainly insoluble in vivo(Cremers et al,

2010), it is possible that the presence of the Hsp33(M172S) in protein aggregates represents a bona

fide chaperone-substrate interaction.

33ºC

22ºC

A

70kDa -

55 -

35 -

25 -

17 -

11 -

- Hsp33(1-235)

B

Fig. 3: Hsp33(1-235) mutant does not support bacterial growth in the absence of DnaK

and TF. (A) In vivo complementation of MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

temperature-sensitive phenotype by pSE-Hsp33 and pSE-Hsp33(1-235) in the presence of

50 µM IPTG, using the procedure described in Fig. 1. (B) Coomassie-stained SDS-PAGE

showing the steady state expression of Hsp33(1-235) at 22°C in strain MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

after 6 h induction at 22°C in the presence of 50 µM IPTG.

Page 55: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

54

The monomeric and redox-regulated Hsp33(Q151E) mutant displayed a sharply different

behavior. It turned out that even at the permissive temperature of growth Hsp33(Q151E) was highly

toxic to bacterial growth at all temperatures tested in the MC4100 Δtig ΔdnaKdnaJ mutant

background when induced(Fig. 1A). In sharp contrast to its deleterious effect on bacterial growth,

monomeric Hsp33(Q151E) fully prevented the accumulation of protein aggregates at the non-

permissive temperature of 37°C, as well as, or even better than Hsp33 wild-type (Fig. 1B). These

findings raise the possibility that the monomeric Hsp33 species, which is believed to be transiently

populated during the Hsp33 activation cascade process and which shows strong preference for

slowly unfolding substrates in vitro(Graf et al, 2004; Jakob et al, 2000), may exhibit robust

substrate binding properties in the absence of the TF/DnaK pathway.

2- Hsp33 function is critical in the absence of both TF and DnaK

The suppression of both the Δtig ΔdnaKdnaJ temperature-sensitive growth phenotype and

the intracellular protein folding defects following Hsp33 overproduction reveal an important

functional cooperation among these three major chaperones. To get additional biological clues for

such interplay we examined the phenotypes of various mutational combinations among these

chaperone genes in the MC4100 strain background. A phenotypic analysis of the double Δtig ΔhslO

and ΔdnaKdnaJ ΔhslO mutants revealed only minor growth differences compared to their

respective single mutant parents, i.e., the double ΔdnaKdnaJ ΔhslO mutant was more temperature-

sensitive for growth at 40°C than its isogenic single ΔdnaKdnaJ mutant (Fig. 4).

Page 56: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

55

30ºC

37ºC

40ºC

43ºC

IPTG

50µM

Fig. 4: Comparative growth of the MC4100 Δtig, Δtig ΔhslO,ΔdnaKdnaJ,ΔdnaKdnaJ ΔhslO

chaperone mutants. Mid-log phase cultures of (i) MC4100 ∆tig::CmR

, ∆tig::CmR

ΔhslO,∆dnaKdnaJ::KanR

,∆dnaKdnaJ::KanR

ΔhslO wereserial diluted and spotted on LB agar

plates and (ii)MC4100 ∆dnaKdnaJ::KanR

ΔhslO complemented with p29SEN vector or with

p29-Hsp33 were serial diluted and spotted on LB ampicillin agar plates supplemented with

50µM IPTG at different temperatures.

Page 57: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

56

Next, we tested the viability of the triple Δtig ΔdnaKdnaJ ΔhslO mutant compared to

itsisogenic Δtig ΔdnaKdnaJ mutant parent. To do so, we used the same methodology as we

previously described for the construction of the Δtig ΔdnaKdnaJ chaperone mutant (Genevaux et al,

2004; Teter et al, 1999). Specifically, we first investigated the co-transduction frequency between

the ΔdnaKdnaJ::KanR mutant allele and itsnearby ~40% linked thr::Tet

R resistance marker using

either MC4100 Δtig or MC4100 Δtig ΔhslO asrecipient strains (Fig. 5A). As anticipated from our

previous work, the ΔdnaKdnaJ mutation was efficiently transduced into the Δtig single mutant with

the expected 40% co-transduction frequency. In contrast, the occasional ΔdnaKdnaJ::KanRthr::Tet

R

co-transductants formed extremely small colonies and, in addition, appeared at a ten-fold lower

frequency (4% instead compared to the expected ~40%) when the Δtig ΔhslO strain was used as the

recipient. Most likely, the few, slowly growing transductants are due to the continuous

accumulation of unknown extragenic suppressors, which enable the occasional survival of the Δtig

ΔdnaKdnaJ ΔhslO triple mutant.

To further demonstrate such a synergy among the various chaperones, we took advantage of

the phenotype exhibited by null mutations in the dnaJ gene (the main DnaK co-chaperone), which

exhibit a significantly less severe growth phenotype defect due to the presence of the djlA and cbpA

genes, known to code for two additional DnaJ-like family members, capable of partially replacing

DnaJ function (Genevaux et al, 2007; Genevaux et al, 2004). We previously showed that the double

tigdnaJ mutant is temperature-sensitive for growth above 34°C in a strictly DnaK-dependent

manner (Genevaux et al, 2004). Based on these earlier results, we introduced the dnaJ::Tn10-42

mutation into either the MC4100 Δtig or MC4100 Δtig ΔhslO mutant backgroundsat 30°C and

subsequently compared their bacterial growth phenotypes at various temperatures (Fig. 5B). In

agreement with the synthetic lethality observed in the absence of DnaK (Fig. 5A), the triple Δtig

ΔhslO dnaJ mutant growth was shown to be significantly more temperature-sensitive than its

isogenic Δtig ΔdnaJ double mutant parent. As expected, the colony-forming ability of this triple

mutant was fully restored following Hsp33 expression from a plasmid (Fig. 5B). The absence of TF,

Hsp33 or DnaJ protein antigens in the corresponding null mutant strains was confirmed by western

blot analysis (Fig. 5C). Taken together, our above data highlight for the first time the essential

functional interplay between the TF, DnaKJE and Hsp33 chaperones in vivo.

Page 58: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

57

Transductants

Recipient strains TetR KanR

MC4100 ∆tig 283 113 (39%)

MC4100 ∆tig ∆hslO 277 13* (4%)

A

B

33ºC

30ºC

37ºC

30ºC

-TF

C

-DnaJ

-Hsp33

Fig. 5: Hsp33 is essential for bacterial viability in the absence of DnaK and TF. (A)

Synthetic lethality among the dnaK, tig and hslO genes was analyzed by P1-mediated

transduction experiments. Transductants were first selected on tetracycline plates following a

two days incubation at 22°C (the permissive temperature for the tig dnaK doublemutant) and

subsequently tested for possession of the kanamycin resistance carried by the linked

ΔdnaKdnaJ::KanR

mutant allele. The co-transduction frequencies of the KanR

marker are

given as percentage of the total number of TetR

transductants. The asterisk indicates the

formation of very small colonies. (B) Synergistic effect of the tig,dnaJ and hslO mutations on

bacterial growth. Mid-log phase cultures of either (i) MC4100 ΔtigdnaJ::Tn10-42 and

MC4100 ΔtigΔhslOdnaJ::Tn10-42, or (ii) MC4100 dnaJ::Tn10-42ΔtigΔhslO containing

p29SEN-Hsp33 were serially diluted ten-fold and spotted on LB agar plates supplemented

with 500 µM IPTG. (C) Whole-cell extracts of MC4100, MC4100 ΔtigdnaJ::Tn10-42 and

MC4100 ΔtigΔhslOdnaJ::Tn10-42 were separated by SDS-PAGE and analyzed by western

blot using anti-TF, -DnaJ and -Hsp33 antibodies.

Page 59: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

58

3- Hsp33 specifically interacts with EF-Tu

The genetic suppression results suggested that, as observed for SecB and GroESL

(Genevaux et al, 2004; Ullers et al, 2004), Hsp33 could bind de novo substrates of TF and DnaKJE

and efficiently prevent their aggregation. However, in contrast to SecB, in vitro crosslinking

experiments did not reveal any co-translational interaction with our model substrate RpoB150,

previously shown to interact with TF, DnaK and SecB, and to aggregate in the absence of TF and

DnaK (Fig. 6;(Ullers et al, 2004)). We next proceeded to search for Hsp33 interacting proteins in

four isogenic strains, namely, MC4100 wild type and its Δtig, ΔdnaKdnaJ and Δtig ΔdnaKdnaJ

mutant derivatives. In these strains, in vivo pull-down experiments were carried out using over-

expressed Flag-tagged Hsp33 as bait (Fig. 7A). An analogous pull-down experiment with a lysate

expressing untagged Hsp33 is shown as a control in Fig. 6. We found that one major Hsp33

interacting partner could be detected in all four genetic backgrounds tested, which was identified by

mass spectrometry analysis to be the essential elongation factor EF-Tu, a key regulator of the

polypeptide chain elongation cycle during the translation process (Fig. 7A; (Thompson et al, 1986)).

This finding suggested that the specific interaction between EF-Tu and Hsp33 might be relevant for

the suppression of the Δtig ΔdnaKdnaJ mutant phenotypes by Hsp33.

Page 60: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

59

X-link IP

250 kDa -

130 -

100 -

70 -

55 -

35 -

25 -

17 -

Hsp33Flag

EF-Tu

**

*

BAA

30-

46-

69-

97-

14-

RpoB150

Fig. 6: Hsp33 interactor. (A) Search for co-translational interaction between ribosome-

associated nascent chain of RpoB150 and Hsp33-FLAG using in vitro translation of RpoB150

transcripts in a Hsp33-FLAG enriched lysate of MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

.

Crosslinking was performed with 1mM of BS3 cross-linker or with water (H

2O) as a

control.Complexes were purified and separated directly or immunoprecipitated using an

antibody anti-FLAG and migrated on SDS-PAGE as described by Ullers et al, (2004). The

bracket indicates the expected area for a RpoB150-Hsp33-FLAG crosslinking product. (B)Pull-

down control with untagged (Hsp33) or tagged Hsp33-Flag or Hsp33(Q151E)-Flag expressed in

MC4100, as described in Fig. 7A. Asterisks represent nonspecific interactions with the Anti-Flag

M2-Agarose resin observed in the absence of tagged Hsp33.

Page 61: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

60

Next, we monitored the endogenous EF-Tu levels in response to Hsp33 over-expression in

the Δtig ΔdnaKdnaJ mutant. In this case, the Hsp33(Y12E) and Hsp33(Q151E) were used as

inactive and hyper-active controls respectively, as deduced from the results shown in Fig. 1. We

found that the endogenous EF-Tu levels rapidly diminished following Hsp33 over-expression,

especially at the non-permissive temperature of 37°C (Fig. 7B). A prolonged over-expression of

Hsp33 at 22°C also led to a decrease of EF-Tu (Fig. 8). In addition, the effect on EF-Tu was

exacerbated in the presence of the constitutively monomeric and redox-regulated Hsp33(Q151E)

mutant (Fig. 7B), which also efficiently binds EF-Tu as shown in Fig. 6. This last result suggests

that distinct intermediates populating the multistep activation cycle of Hsp33 could possess unique

functions in vivo(Akhtar et al, 2004; Chi et al, 2011; Graf et al, 2004). In contrast, and as expected,

the Hsp33(Y12E) mutant was completely non-functional, behaving like the plasmid vector control

(Fig. 7B).

B

250 -

130 -

100 -

70 -

55 -

35 -

25 -

17 -

Hsp33

EF-Tu

-EF-Tu

250 -

130 -

100 -

70 -

55 -

35 -

25 -

17 -

Hsp33Flag

EF-Tu

A

Fig. 7: Hsp33 specifically interacts with EF-Tu. (A) Identification of the Hsp33 interacting substrates by

pull-down analysis using an Hsp33-FLAG version expressed in either MC4100, or MC4100 ∆tig::CmR

, or

MC4100 ΔdnaKdnaJ::KanR

or MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

at 30°C. The main co-precipitated

protein obtained in all four strains was EF-Tu as identified by a mass spectrometric analysis. (B)

Endogenous levels of EF-Tu in MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

following over-expression of Hsp33

wild type or its mutant derivatives Hsp33(Y12E) and Hsp33(Q151E). Cultures of MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

transformed with the various pSE380-based constructs were grown in LB ampicillin,

supplemented with 0.4% glucose at 22°C to an OD600

of 0.3. IPTG was then added at 500 µM for 30 min at

22°C for pre-induction and cultures were transferred to either 22°, or 30° or 37°C for 1 h and 30 min. Whole

cell extracts were prepared, separated by SDS-PAGE and stained with Coomassie blue. The characterization

of the EF-Tu protein was confirmed by the western blot analysis shown at the bottom of the panel.

Page 62: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

61

Although at this stage we cannot completely rule out the possibility that Hsp33, as a

chaperone, directly prevents protein aggregation in the absence of TF and DnaK, our results

strongly suggest that modulation of the rate of polypeptide chain elongation by Hsp33 might be

responsible for bacterial survival. Our in vivo pull-down experiments described above never

revealed a major difference in the qualitative nature of the Hsp33 interactor pattern. Nevertheless,

we reproducibly noticed that the EF-Tu/Hsp33 ratio was higher in the wild type and the Δtig

mutantthan for the ΔdnaKdnaJ or Δtig ΔdnaKdnaJ isogenic mutant strains (Fig. 7A). Consequently,

we tested whether the effect of Hsp33 on EF-Tu was different in the absence of TF, DnaK or both.

To do so, we over-expressed Hsp33 in the four isogenic strains and monitored the EF-Tu levels in

whole cell extracts. The results presented in Fig. 9A show that upon Hsp33 over-expression, the EF-

Tu levels are not significantly affected in the wild-type strain, besides the slight decrease in EF-Tu

observed for the single Δtig mutant at 30°C and 37°C. In contrast, the EF-Tu intracellular levels

250 kDa -

130 -

100 -

70 -

55 -

35 -

25 -

17 -

MC4100 Δtig ΔdnaKdnaJ

vector Hsp33

Hsp33

EF-Tu

time (min)

Fig. 8: Steady state levels of EF-Tu following a prolonged expression of Hsp33 in the

absence of DnaK and TF. Overnight cultures of MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

transformed with pSE380 vector or with pSE-Hsp33 grown in LB ampicillin glucose

0.4% at 22°C were diluted 1/50 and grown until OD600

0.3. IPTG 500µM was then added

for 30 min at 22°C and strains were transferred at 30°C for 0, 90 min, 120 min, 150 min,

180 min. Whole cell extracts were prepared and separated on SDS-PAGE, and proteins

were stained with Coomassie Blue.

Page 63: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

62

dropped very rapidly when Hsp33 was over-expressed in the ΔdnaKdnaJ mutant, even at 30°C. The

expression of Hsp33 in the Δtig ΔdnaKdnaJ mutant resulted in an intermediate behavior. Note that

the Hsp33 over-expression levels were comparable in all instances (Fig. 9A). The results presented

in Fig. 9B suggest that the diminution of the endogenous EF-Tu directly correlates with bacterial

growth inhibition at the otherwise permissive temperature of 22°C, coupled with a marked toxicity

of Hsp33 in the ΔdnaKdnaJ mutant.

-RpoB150

C

*

100µM

IPTG

22ºC

B

-

- EF-Tu

A

130 -

100 -

70 -

55 -

35 -

25 -

17 -

Hsp33

Fig. 9: Hsp33-mediated control of EF-

Tu in the absence of DnaK. (A)

Endogenous EF-Tu levels in MC4100,

MC4100 ∆tig::CmR

, MC4100

ΔdnaKdnaJ::KanR

or MC4100

∆tig::CmR

∆dnaKdnaJ::KanR

following

Hsp33 over-expression were analyzed as

described in the legend to Fig. 7B. Arrows

indicate the position of EF-Tu in whole

cell extracts. (B) Over-expression of

Hsp33 (pSE-Hsp33) in MC4100 and its

mutant derivatives. Bacterial cultures

grown at 22°C were serially diluted ten-

fold and spotted on LB ampicillin agar

plates at 22°C with or without IPTG 100

µM. The letter v stands for the vector and

the number 33 stands for the Hsp33-

carrying plasmid. (C) In vitro translation

of the first 150 amino acids of RpoB using

cell- and membrane-free extracts of

MC4100 ΔdnaKdnaJ::KanR

and MC4100

∆tig::CmR

∆dnaKdnaJ::KanR

mutants over-

expressing Hsp33-FLAG. After in vitro

translation, radiolabeled samples were

resuspended in loading buffer and

separated by a SDS/15% PAGE. The

asterisk indicates the position of the

truncated RpoB150 polypeptide (left lane).

The position of the full length RpoB150 is

indicated as RpoB150 (right lane).

Page 64: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

63

These findings suggest that in the absence of TF and DnaK, over-expressed Hsp33 may

target EF-Tu for degradation by proteases. Moreover, the effect on EF-Tu was exacerbated in the

ΔdnaKdnaJ mutant, thus suggesting that TF may also facilitate interaction of EF-Tu with Hsp33,

leading to its subsequent degradation. We first examined whether Hsp33 over-expression

differentially affects polypeptide chain elongation in vitro. To do so, we prepared translation lysates

from both ΔdnaKdnaJ and Δtig ΔdnaKdnaJ mutant strains over-expressing Hsp33, and

subsequently compared their ability to support translation of the RpoB150 mRNA in vitro(Ullers et

al, 2004). In agreement with our previous work, we found that cell lysates obtained from the Δtig

ΔdnaKdnaJ mutant strain indeed efficiently translated RpoB150 mRNA, as judged by the presence

of labeled, full length RpoB150 nascent chains (Fig. 9C;(Ullers et al, 2004)). In sharp contrast,

lysates prepared from the ΔdnaKdnaJ mutant strain expressing Hsp33 were not capable of

translating the RpoB150 mRNA to completion, as judged by the absence of the full length RpoB150

nascent chain, with concomitant appearance of a putative short truncated RpoB150 translation

product (marked with an asterisk; left lane of Fig. 9C). These results are in agreement with the

observed dramatic decrease in the levels of endogenous EF-Tu in the absence of DnaK with the

simultaneous overproduction of Hsp33 and the resulting strong toxicity on bacterial growth(Fig. 9A

and B).

4- Hsp33 triggers EF-Tu degradation by the stress protease Lon

Our results suggest that in the absence of DnaK, and to lesser extent in the absence of both

DnaK and TF, the chaperone Hsp33 specifically binds and delivers EF-Tu to yet unknown

protease(s). In order to identify such protease(s), we took advantage of the strong DnaK-dependent

toxicity of Hsp33 observed in Fig. 9B. We reasoned that mutation in the specific protease might

abolish Hsp33-dependent decrease of endogenous EF-Tu and the resulting toxicity. Therefore, we

combined single deletion of either Δlon, ΔclpP or ΔclpQ major cytoplasmic AAA+ proteases(Sauer

& Baker, 2011) with the ΔdnaKdnaJ mutation at 30°C, over-expressed Hsp33 and monitored its

effect on bacterial growth and EF-Tu. The results presented in Fig. 10A clearly show that in

contrast to ΔclpP and ΔclpQ, the Δlon mutation efficiently suppresses Hsp33 toxicity and restores

endogenous EF-Tu close to wild-type level. Yet, a low amount of EF-Tu was also recovered in the

absence of clpP, suggesting some overlap between Lon and ClpP for this substrate (Fig. 10A,

bottom).

To ensure that the decreased EF-Tu levels were due to EF-Tu degradation and not to an

effect on transcription, the stimulation of EF-Tu degradation by over-expressed Hsp33 was further

demonstrated using pulse chase experiments followed by immunoprecipitation of EF-Tu in the

Page 65: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

64

ΔdnaKdnaJ strain expressing Hsp33. The results presented in Fig. 10B show that indeed, Hsp33

significantly accelerates EF-Tu degradation in the absence of DnaK, suggesting that Hsp33

maintains EF-Tu in a conformation competent for degradation.

-EF-Tu

ΔdnaKdnaJ

30ºC

500µM

IPTG

-

-

A

B

ΔhslO ΔdnaKdnaJ

+Hsp33

-

Chase(min) 0 60 120

EF-Tu

Fig. 10: Hsp33 stimulates Lon protease-

mediated degradation of EF-Tu. (A)

Cultures of MC4100 ΔdnaKdnaJ::KanR

,

MC4100 Δlon ΔdnaKdnaJ::KanR

, MC4100

ΔclpP::CmR

ΔdnaKdnaJ::KanR

and MC4100

ΔclpQ::TetR

ΔdnaKdnaJ::KanR

transformed

with pSE380 vector (indicated as v) or pSE-

Hsp33 (indicated as 33), were serially diluted

ten-fold and spotted on LB ampicillin agar

plates with or without 500 µM IPTG inducer

at 30°C. Steady state levels of EF-Tu in the

corresponding strains were analyzed by

western blot analysis using liquid cultures

following a 2 h incubation in the presence of

500 µM IPTG inducer at 30°C. (B) The

kinetics of EF-Tu degradation in vivo in

MC4100 ∆hslO ∆dnaKdnaJ::KanR

bacteria

transformed with either the pSE380 vector or

pSE-Hsp33 was analyzed by first labeling

with 35

S-methionine, followed by a 0, or 60

or 120 min chase with excess, unlabeled

methionine and co-immunoprecipitation.

Page 66: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

65

5- EF-Tu inhibition helps bacterial growth in the absence of TF and DnaK

The proposed control of polypeptide chain elongation by a network of chaperones and

proteases suggests that the suppression by Hsp33 in the absence of TF and DnaK is linked to EF-Tu

degradation and not to the previously observed in vitro chaperone activity of EF-Tu(Caldas et al,

1998). In agreement with such a hypothesis, EF-Tu over-expression does not rescue the growth

defect of the MC4100 Δtig ΔdnaKdnaJ mutant strain (Fig. 11A). As a control, the steady state level

of plasmid-encoded EF-Tu is shown in Fig. 12. We next examined whether Hsp33-mediated

degradation of EF-Tu by Lon was responsible for the suppression in the absence of TF and DnaKJ.

We expressed Hsp33 both in the Δtig dnaJ and in the Δtig Δlon dnaJ mutants, and compared

suppression of the temperature-sensitive phenotype by Hsp33. Note that as observed for MC4100

Δtig ΔdnaKdnaJ, over-expression of Hsp33 in the less sensitive MC4100 ΔtigdnaJ mutant strain

similarly stimulated EF-Tu degradation by Lon at high temperature of growth (Fig. 12).

Remarkably, the results presented in Fig. 11B clearly show that in contrast with the expected

suppression observed for the Δtig dnaJ (lon+) double mutant, Hsp33 was not capable of rescuing

bacterial growth in the absence of Lon (Fig. 11B). These results strongly suggest that control of the

endogenous EF-Tu levels is critical when the TF/DnaKJE pathway is compromised.

Next, we took advantage of the HipA toxin of the HipAB toxin-antitoxin module from E.

coli, known to bind to and inhibit EF-Tu by phosphorylation at residue Thr382, thus compromising

polypeptide chain elongation(Hansen et al, 2012; Schumacher et al, 2009). We first asked whether

HipA over-expression could mimic Hsp33-mediated control of EF-Tu in the absence of both TF and

DnaK. To do so, gene encoding HipA was cloned in a plasmid under the control of the tightly

regulated araBADp promoter (to avoid growth inhibition by the toxin) and expressed in the

sensitive Δtig ΔdnaKdnaJ mutant. As expected for a toxin affecting translation, HipA expression

significantly slowed down bacterial growth at the permissive temperature of 22°C (Fig. 11C). In

contrast, at the non-permissive temperature of 37°C, a mild over-expression of HipA efficiently

rescued bacterial growth and prevented protein aggregation in the absence of both TF and DnaK

(Fig. 11C and D). These results indicate that a partial inactivation of EF-Tu function may be

beneficial to bacterial survival in the absence of the major TF and DnaK chaperones and highlights

a possible role for the toxin-antitoxin system in controlling the rate of protein synthesis in response

to protein aggregation (see Discussion below).

Page 67: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

66

C

HipA

22°C 33°C

Δtig ΔdnaKdnaJ

- +

37°C

- + - +

30°C

37°C

A

Hsp33-

B

D

WCE Agg.

130 -

100 -

70 -

55 -

35 -

25 -

17 -

37°C

50050-IPTG

22°C

33°C

Fig. 11: EF-Tu inactivation facilitates growth and protein folding in the absence of TF and DnaK

chaperones. (A) EF-Tu over-expression in MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

. Cultures of

MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

transformed with p29SEN or with p29-EF-Tu were grown at

22°C, serially diluted ten-fold and spotted on LB ampicillin agar plates with or without IPTG and

incubated at 22° or 33°C. (B) Cultures of MC4100 Δtig::CmR

dnaJ::Tn10-42 or MC4100

Δtig::CmR

dnaJ::Tn10-42 Δlon::KanR

transformed with either p29SEN (indicated as v) or with p29-

Hsp33 (indicated as 33) were grown in LB ampicillin, supplemented with glucose 0.4% at 30°C,

serially diluted ten-fold and spotted on LB ampicillin agar plates with or without IPTG 50 µM at 30°

or 37°C. Protein levels were analyzed by western blot analysis of the extracts following a 2 h

induction with IPTG 50 µM at 30°C. The genetic background of each strain used is shown on top of

each panel. (C) TheHipA toxin efficiently rescues bacterial growth in the absence of TF and DnaK.

Strain W3110 Δtig::CmR

ΔdnaKdnaJ::KanR

pBAD-HipA was grown without (-) or with (+) 0.5% of

L-arabinose inducer at the indicated temperatures. (D) Prevention of protein aggregation by HipA

toxin expressed from the pBAD-HipA plasmid in strain W3110 Δtig::CmR

ΔdnaKdnaJ::KanR

. HipA

expression was induced with 0.5% L-arabinose for 6 h at 22°C followed by 1 h 30 min at 33°C. Whole

cell extracts and aggregates were separated by SDS-PAGE and stained with Coomassie blue.

Page 68: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

67

EF-Tu EF-Tu

BA

Fig. 12: EF-Tu expression. (A) EF-Tu over-expression from plasmid p29-EF-Tu in

MC4100 ∆tig::CmR

∆dnaKdnaJ::KanR

strain was induced overnight with 500µM IPTG at

22°C. EF-Tu was revealed by western blot using anti-EF-Tu antibody. (B) EF-Tu

endogenous levels following Hsp33 over-expression in the absence of either TF/DnaJ, or

TF/DnaJ/Lon. Overnight cultures of MC4100 ∆tig dnaJ (lon+

) and MC4100 ∆tig dnaJ

∆lon transformed with pSE-Hsp33 grown in LB ampicillin glucose 0.4% at 30°C were

diluted 1/50 and grown until OD600

0.3. IPTG 500µM was then added for 30 min at 30°C

and strains were transferred at 43°C for 3 h. Whole cell extracts were prepared and

separated on SDS-PAGE, and EF-Tu was revealed by western blot using anti-EF-Tu

antibody.

Page 69: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

68

DISCUSSION

Page 70: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

69

DISCUSSION

The heat shock protein Hsp33 is a highly specialized redox-regulated molecular chaperone, which is

activated by a combination of both oxidative stress and protein unfolding or aggregation (Kumsta &

Jakob, 2009). In this work, we propose that Hsp33 can also perform key, house-keeping cellular

functions in the absence of exogenous stressors as part of the network of stress chaperones and

proteases in E. coli. This conclusion was reached by demonstrating that over-expression of Hsp33

can efficiently support bacterial growth and prevent accumulation of protein aggregates in the

absence of TF and DnaK. However, in contrast with other chaperones, such as SecB and GroESL

(Genevaux et al, 2004; Ullers et al, 2004; Vorderwulbecke et al, 2004), suppression by Hsp33 does

not seem to rely on its general chaperone function. Instead, it appears that Hsp33 efficiently binds to

the essential elongation factor EF-Tu and specifically targets it for degradation by the Lon protease.

The subsequent decrease in endogenous EF-Tu level may result in a decrease of the intracellular

rate of nascent polypeptide synthesis, thus minimizing the need for action by downstream

chaperones TF and DnaKJE.

Although we do not know yet about the relevance of such pathway under physiological

conditions where all the chaperones are present, our results point toward the existence of an

intricate network of stress chaperones and proteases that result in an inhibition of the rate of

translation in response to severe protein aggregation. It is known that under stress conditions that

affect protein folding, several major, stress-induced chaperones, including DnaK, are recruited to

the accumulating protein aggregates (Winkler et al, 2010). Importantly, such a recruitment of DnaK

to protein aggregates results in the rapid stabilization of the heat shock sigma factor σ32

, thus

leading to a prolonged induction of heat shock proteins, including Hsp33 and Lon. Note that in our

case, both the induction of heat shock proteins and the aggregation-prone conditions are, at least in

part, artificially recreated by the introduction of the tig dnaKdnaJ mutations (Genevaux et al, 2004).

Yet, in the absence of these dominant chaperones, we cannot exclude that induction of the stress

response or accumulation of extragenic suppressors that help bacterial growth without TF and

DnaK could force the bacteria to use pathways that are not utilized under normal conditions (Bukau

& Walker, 1990; Kramer et al, 2004b). In our working model, the increased recruitment of the

major molecular chaperones to preexisting aggregates and newly-synthesized proteins rapidly

overpowers the cellular chaperone capacity available for de novo protein folding. This, in turn,

results in a signal that triggers a transient inhibition of the rate of translation and a subsequent

reduction in the intracellular levels of newly-synthesized polypeptides that normally necessitate

molecular chaperones for their proper folding (Hartl et al, 2011). Such a possible mechanism is

supported by previous studies showing that slowing down translation facilitates de novo protein

Page 71: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

70

folding in E. coli(Agashe et al, 2004; Siller et al, 2010) and in mammalian cells, as shown recently

(Meriin et al, 2012).

If such an intricate control of translation rate by the availability of stress chaperones and

proteases indeed exists, then the results presented in this work clearly point to the elongation factor

EF-Tu as one of the major targets of such an inhibition. Interestingly, in addition to the Hsp33/EF-

Tu complex demonstrated in this study, an interaction between EF-Tu and Lon has also been found

in vivo(Butland et al, 2005), further supporting the proposed interplay among chaperones and

proteases. However, it remains to be determined whether EF-Tu is the sole protein directly targeted

for degradation by the Hsp33/Lon pathway and whether other minor interactions that our

experimental procedures are missing could also participate.

It is known that EF-Tu adopts several conformations and populates various intermediate

stages during its functional cycle in polypeptide chain elongation (Agirrezabala & Frank, 2009). At

this time, the actual conformational state(s) of EF-Tu that is recognized by Hsp33 and Lon remains

unknown. Intriguingly, in contrast to our finding that in E. coli Hsp33 stimulates EF-Tu degradation

by Lon, it was recently shown in V. cholerae that Hsp33 was instead required to maintain EF-Tu at

cellular levels sufficient to allow bacterial growth at high temperature under aerobic conditions

(Wholey & Jakob, 2012). In this case, Hsp33 stabilized the highly sensitive V. cholerae EF-Tu

protein from oxidative protein degradation in vivo. Interestingly, the E. coli EF-Tu protein was not

affected under the same conditions (Wholey & Jakob, 2012). The differential results seen with

different bacteria strongly suggest that although the proposed network controlling EF-Tu

intracellular levels is conserved, the overall outcome may differ depending on the particular

organism and/or stress conditions employed.

The fact that degradation of EF-Tu by Hsp33 and Lon predominantly occurs when DnaK is

absent suggests that under normal growth conditions DnaK prevents EF-Tu degradation, either by

directly interacting with EF-Tu or indirectly by inhibiting the σ32

-dependent synthesis of Lon. In

contrast with such a protective effect of DnaK, we found that the presence of TF significantly

stimulates EF-Tu degradation, indicating that TF directly or indirectly facilitates the interaction of

EF-Tu with Hsp33 and/or its subsequent recognition by Lon. Such a hypothesis is strongly

supported (i) by a recent study demonstrating that in contrast with DnaK, the ribosome-bound TF

chaperone can unfold substrates and facilitate their degradation in vitro(Hoffmann et al, 2012), and

(ii) by the synthetic lethality observed between the tig, dnaKdnaJ and hslO mutations (Fig. 5A).

How does Hsp33 contribute to such a network in the absence of oxidative stressors? One

possibility is that Hsp33 is only activated by increasing levels of reactive oxygen species (ROS)

stimulated by the severe protein homeostasis collapse induced by the lack of both TF and DnaK

Page 72: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

71

major chaperones (Calloni et al, 2012). However, this seems unlikely because (i) Hsp33 efficiently

binds EF-Tu in the wild type E. coli strain as well, and (ii) EF-Tu is much more efficiently degraded

in the dnaKdnaJ mutant than in the triple tig dnaKdnaJ mutant where proteostasis breakdown is the

most severe (Deuerling et al, 1999; Genevaux et al, 2004; Teter et al, 1999). Therefore, we propose

that a significant fraction of endogenous Hsp33 may be active and physiologically relevant even in

the absence of oxidative stress. This is supported by the synergistic effects observed among the tig,

dnaK (or dnaJ)and hslO mutations. However, the redox and the oligomeric status (monomeric,

dimeric or oligomeric) of cellular Hsp33 that is capable of performing such a function in vivo

remains to be determined (Akhtar et al, 2004; Chi et al, 2011; Graf et al, 2004). In addition, it is not

yet established whether Hsp33 induces EF-Tu degradation under physiological conditions where all

the chaperones are present. As stated above, Hsp33 is activated by oxidative protein unfolding

(Chuang & Blattner, 1993; Jakob et al, 1999), a condition that is known to impede DnaK functions

(Hoffmann et al, 2004). Under such extreme stress condition, quality control of EF-Tu by activated

Hsp33 and Lon could thus become a key to bacterial survival (Fig. 13). Further studies are needed

to elucidate such possible interplay under severe oxidative conditions.

Analogous to the newly identified cooperation between Hsp33 and Lon in controlling of EF-

Tu, over-expression of the HipA toxin of the chromosomally-encoded type-II HipAB toxin-

antitoxin system can also suppress both the growth defect and the accumulation of protein

aggregates in the absence of TF and DnaK. Toxin-antitoxin modules are two-component systems

composed of a stable toxin that forms an inactive complex with its less stable cognate antitoxin. In

response to specific stress conditions the unstable antitoxin is generally degraded by stress proteases

and the thusly free active toxin subsequently targets important cellular processes such as DNA

replication or protein synthesis, resulting in growth inhibition and eventual cell death (Gerdes et al,

2005; Moll & Engelberg-Kulka, 2012; Van Melderen & De Bast, 2009; Yamaguchi et al, 2011) .

Recent work has shown that the E. coli toxin HipA is a protein kinase belonging to the Tor family,

which specifically inhibits EF-Tu and blocks translation (Correia et al, 2006; Schumacher et al,

2009). In agreement with such an activity, over-expression of HipA slows down E. coli growth and

increases antibiotic tolerance and persistence (reviewed in (Lewis, 2010)). Although it is well

established that many stress-responsive toxin-antitoxin systems affect translation in bacteria, the

finding that an active toxin can partially bypass major molecular chaperone requirements in vivo is

novel. An attractive hypothesis is that severe protein aggregation may trigger the activation of

stress-responsive toxin-antitoxin systems in order to control the level of protein synthesis, thus

relieving the overwhelmed downstream network of molecular chaperones until normal conditions

resume. In agreement with this hypothesis, four other well-characterized antitoxins present in E.

Page 73: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

72

coli, namely MazE, RelB, MqsA and DinJ, whose respective cognate toxins MazF, RelE MqsR, and

YafQ are known to affect protein synthesis, have been recently shown to be bona fide DnaK

substrates in vivo(Calloni et al, 2012). Clearly, further work is needed to elucidate such a possible

role for chromosomally-encoded toxin-antitoxin systems in bacteria.

EF-Tu degradation

Heat Shock ResponseSynthesis of HSPs

(incl. Lon and Hsp33)

Protein aggregation

Recruitment ofDnaKJE and GroESL

Slowing down of translation (decreased protein load on

chaperones)

STRESS

Adaptation to stress

Fig. 13: Hypothetical model for Hsp33/Lon-mediated degradation of EF-Tu in response to

severe protein aggregation. (1), Denaturing stress induces protein aggregation; (2), DnaKJE and

GroESL are rapidly recruited to protein aggregates; (3), titration of both chaperone machines by

aggregates stabilizes the heat-shock sigma factor σ32

, which subsequently induces the synthesis of

heat-shock proteins (HSPs), including Hsp33 and Lon; (4), Hsp33 and Lon stimulate EF-Tu

degradation, slowing down de novo protein synthesis; (5), the reduced load of newly synthesized

proteins on the downstream chaperones contributes to bacterial survival until normal conditions

resume.

Page 74: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

73

REFERENCES

Page 75: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

74

REFERENCES

Acebron SP, Fernandez-Saiz V, Taneva SG, Moro F, Muga A (2008) DnaJ recruits DnaK to protein

aggregates. J Biol Chem283: 1381-1390

Acebron SP, Martin I, del Castillo U, Moro F, Muga A (2009) DnaK-mediated association of ClpB to protein

aggregates. A bichaperone network at the aggregate surface. FEBS Lett583: 2991-2996

Agashe VR, Guha S, Chang HC, Genevaux P, Hayer-Hartl M, Stemp M, Georgopoulos C, Hartl FU, Barral

JM (2004) Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the

expense of folding speed. Cell117: 199-209

Agirrezabala X, Frank J (2009) Elongation in translation as a dynamic interaction among the ribosome,

tRNA, and elongation factors EF-G and EF-Tu. Q Rev Biophys42: 159-200

Akhtar MW, Srinivas V, Raman B, Ramakrishna T, Inobe T, Maki K, Arai M, Kuwajima K, Rao Ch M

(2004) Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-

ray scattering (SAXS) analysis. J Biol Chem279: 55760-55769

Akoev V, Gogol EP, Barnett ME, Zolkiewski M (2004) Nucleotide-induced switch in oligomerization of the

AAA+ ATPase ClpB. Protein Sci13: 567-574

Al Refaii A, Alix JH (2009) Ribosome biogenesis is temperature-dependent and delayed in Escherichia coli

lacking the chaperones DnaK or DnaJ. Mol Microbiol71: 748-762

Allen SP, Polazzi JO, Gierse JK, Easton AM (1992) Two novel heat shock genes encoding proteins produced

in response to heterologous protein expression in Escherichia coli. J Bacteriol174: 6938-6947

Altman E, Kumamoto CA, Emr SD (1991) Heat-shock proteins can substitute for SecB function during

protein export in Escherichia coli. EMBO J10: 239-245

Anderson WF (1969) The effect of tRNA concentration on the rate of protein synthesis. Proc Natl Acad Sci

U S A62: 566-573

Anfinsen CB (1973) Principles that govern the folding of protein chains. Science181: 223-230

Aslund F, Beckwith J (1999) Bridge over troubled waters: sensing stress by disulfide bond formation.

Cell96: 751-753

Bachmann BJ (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev36: 525-557

Ball LA, Kaesberg P (1973) Cleavage of the N-terminal formylmethionine residue from a bacteriophage coat

protein in vitro. J Mol Biol79: 531-537

Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large

ribosomal subunit at 2.4 A resolution. Science289: 905-920

Baram D, Pyetan E, Sittner A, Auerbach-Nevo T, Bashan A, Yonath A (2005) Structure of trigger factor

binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action.

Proc Natl Acad Sci U S A102: 12017-12022

Barbirz S, Jakob U, Glocker MO (2000) Mass spectrometry unravels disulfide bond formation as the

mechanism that activates a molecular chaperone. J Biol Chem275: 18759-18766

Page 76: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

75

Bardwell JC, Craig EA (1988) Ancient heat shock gene is dispensable. J Bacteriol170: 2977-2983

Barnett ME, Nagy M, Kedzierska S, Zolkiewski M (2005) The amino-terminal domain of ClpB supports

binding to strongly aggregated proteins. J Biol Chem280: 34940-34945

Barral JM, Broadley SA, Schaffar G, Hartl FU (2004) Roles of molecular chaperones in protein misfolding

diseases. Semin Cell Dev Biol15: 17-29

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (2007)

CRISPR provides acquired resistance against viruses in prokaryotes. Science315: 1709-1712

Bashan A, Yonath A (2008) Correlating ribosome function with high-resolution structures. Trends

Microbiol16: 326-335

Berks BC (1996) A common export pathway for proteins binding complex redox cofactors? Mol

Microbiol22: 393-404

Berndt U, Oellerer S, Zhang Y, Johnson AE, Rospert S (2009) A signal-anchor sequence stimulates signal

recognition particle binding to ribosomes from inside the exit tunnel. Proc Natl Acad Sci U S A106: 1398-

1403

Bertelsen EB, Chang L, Gestwicki JE, Zuiderweg ER (2009) Solution conformation of wild-type E. coli

Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc Natl Acad Sci U S A106: 8471-8476

Bhushan S, Gartmann M, Halic M, Armache JP, Jarasch A, Mielke T, Berninghausen O, Wilson DN,

Beckmann R (2010) alpha-Helical nascent polypeptide chains visualized within distinct regions of the

ribosomal exit tunnel. Nat Struct Mol Biol17: 313-317

Bingel-Erlenmeyer R, Kohler R, Kramer G, Sandikci A, Antolic S, Maier T, Schaffitzel C, Wiedmann B,

Bukau B, Ban N (2008) A peptide deformylase-ribosome complex reveals mechanism of nascent chain

processing. Nature452: 108-111

Blanchard JL, Wholey WY, Conlon EM, Pomposiello PJ (2007) Rapid changes in gene expression dynamics

in response to superoxide reveal SoxRS-dependent and independent transcriptional networks. Plos One2:

e1186

Blumenthal T, Landers TA, Weber K (1972) Bacteriophage Q replicase contains the protein biosynthesis

elongation factors EF Tu and EF Ts. Proc Natl Acad Sci U S A69: 1313-1317

Bornemann T, Jockel J, Rodnina MV, Wintermeyer W (2008) Signal sequence-independent membrane

targeting of ribosomes containing short nascent peptides within the exit tunnel. Nat Struct Mol Biol15: 494-

499

Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS,

Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science321:

960-964

Bruel N, Castanie-Cornet MP, Cirinesi AM, Koningstein G, Georgopoulos C, Luirink J, Genevaux P (2012)

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the

Major DnaK and Trigger Factor Chaperones. J Biol Chem287: 44435-44446

Bukau B, Horwich AL (1998) The Hsp70 and Hsp60 chaperone machines. Cell92: 351-366

Bukau B, Walker GC (1989) Delta dnaK52 mutants of Escherichia coli have defects in chromosome

segregation and plasmid maintenance at normal growth temperatures. J Bacteriol171: 6030-6038

Page 77: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

76

Bukau B, Walker GC (1990) Mutations altering heat shock specific subunit of RNA polymerase suppress

major cellular defects of E. coli mutants lacking the DnaK chaperone. EMBO J9: 4027-4036

Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B,

Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005) Interaction network containing conserved

and essential protein complexes in Escherichia coli. Nature433: 531-537

Caldas TD, El Yaagoubi A, Richarme G (1998) Chaperone properties of bacterial elongation factor EF-Tu. J

Biol Chem273: 11478-11482

Calloni G, Chen T, Schermann SM, Chang HC, Genevaux P, Agostini F, Tartaglia GG, Hayer-Hartl M, Hartl

FU (2012) DnaK Functions as a Central Hub in the E. coli Chaperone Network. Cell Rep1: 251-264

Casadaban MJ (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia coli

using bacteriophage lambda and Mu. J Mol Biol104: 541-555

Chakraborty K, Chatila M, Sinha J, Shi Q, Poschner BC, Sikor M, Jiang G, Lamb DC, Hartl FU, Hayer-Hartl

M (2010) Chaperonin-catalyzed rescue of kinetically trapped states in protein folding. Cell142: 112-122

Chattopadhyay S, Das B, Dasgupta C (1996) Reactivation of denatured proteins by 23S ribosomal RNA: role

of domain V. Proc Natl Acad Sci U S A93: 8284-8287

Chavancy G, Garel JP (1981) Does quantitative tRNA adaptation to codon content in mRNA optimize the

ribosomal translation efficiency? Proposal for a translation system model. Biochimie63: 187-195

Chi SW, Jeong DG, Woo JR, Lee HS, Park BC, Kim BY, Erikson RL, Ryu SE, Kim SJ (2011) Crystal

structure of constitutively monomeric E. coli Hsp33 mutant with chaperone activity. FEBS Lett585: 664-670

Chiang SM, Schellhorn HE (2012) Regulators of oxidative stress response genes in Escherichia coli and their

functional conservation in bacteria. Arch Biochem Biophys525: 161-169

Chuang SE, Blattner FR (1993) Characterization of twenty-six new heat shock genes of Escherichia coli. J

Bacteriol175: 5242-5252

Clare DK, Vasishtan D, Stagg S, Quispe J, Farr GW, Topf M, Horwich AL, Saibil HR (2012) ATP-triggered

conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin.

Cell149: 113-123

Collier DN, Bankaitis VA, Weiss JB, Bassford PJ, Jr. (1988) The antifolding activity of SecB promotes the

export of the E. coli maltose-binding protein. Cell53: 273-283

Correia FF, D'Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, Koonin EV, Lewis K (2006) Kinase

activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J

Bacteriol188: 8360-8367

Cortazzo P, Cervenansky C, Marin M, Reiss C, Ehrlich R, Deana A (2002) Silent mutations affect in vivo

protein folding in Escherichia coli. Biochem Biophys Res Commun293: 537-541

Cremers CM, Reichmann D, Hausmann J, Ilbert M, Jakob U (2010) Unfolding of metastable linker region is

at the core of Hsp33 activation as a redox-regulated chaperone. J Biol Chem285: 11243-11251

Crombie T, Boyle JP, Coggins JR, Brown AJ (1994) The folding of the bifunctional TRP3 protein in yeast is

influenced by a translational pause which lies in a region of structural divergence with Escherichia coli

indoleglycerol-phosphate synthase. Eur J Biochem226: 657-664

Page 78: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

77

Crooke E, Guthrie B, Lecker S, Lill R, Wickner W (1988) ProOmpA is stabilized for membrane

translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell54: 1003-1011

Crooke E, Wickner W (1987) Trigger factor: a soluble protein that folds pro-OmpA into a membrane-

assembly-competent form. Proc Natl Acad Sci U S A84: 5216-5220

Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12

using PCR products. Proc Natl Acad Sci U S A97: 6640-6645

Defeu Soufo HJ, Reimold C, Linne U, Knust T, Gescher J, Graumann PL (2010) Bacterial translation

elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc Natl Acad Sci U S

A107: 3163-3168

del Castillo U, Alfonso C, Acebron SP, Martos A, Moro F, Rivas G, Muga A (2011) A quantitative analysis

of the effect of nucleotides and the M domain on the association equilibrium of ClpB. Biochemistry50: 1991-

2003

Deuerling E, Patzelt H, Vorderwulbecke S, Rauch T, Kramer G, Schaffitzel E, Mogk A, Schulze-Specking

A, Langen H, Bukau B (2003) Trigger Factor and DnaK possess overlapping substrate pools and binding

specificities. Mol Microbiol47: 1317-1328

Deuerling E, Schulze-Specking A, Tomoyasu T, Mogk A, Bukau B (1999) Trigger factor and DnaK

cooperate in folding of newly synthesized proteins. Nature400: 693-696

Ding H, Demple B (1998) Thiol-mediated disassembly and reassembly of [2Fe-2S] clusters in the redox-

regulated transcription factor SoxR. Biochemistry37: 17280-17286

Dobson CM (2001) The structural basis of protein folding and its links with human disease. Philosophical

transactions of the Royal Society of London Series B, Biological sciences356: 133-145

Dobson CM (2004) Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol15: 3-16

Eisner G, Moser M, Schafer U, Beck K, Muller M (2006) Alternate recruitment of signal recognition particle

and trigger factor to the signal sequence of a growing nascent polypeptide. J Biol Chem281: 7172-7179

Ellis RJ, Minton AP (2003) Cell biology: join the crowd. Nature425: 27-28

Fayet O, Ziegelhoffer T, Georgopoulos C (1989) The groES and groEL heat shock gene products of

Escherichia coli are essential for bacterial growth at all temperatures. J Bacteriol171: 1379-1385

Fedyunin I, Lehnhardt L, Bohmer N, Kaufmann P, Zhang G, Ignatova Z (2012) tRNA concentration fine

tunes protein solubility. FEBS Lett586: 3336-3340

Fekkes P, de Wit JG, van der Wolk JP, Kimsey HH, Kumamoto CA, Driessen AJ (1998) Preprotein transfer

to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to

SecA. Mol Microbiol29: 1179-1190

Ferbitz L, Maier T, Patzelt H, Bukau B, Deuerling E, Ban N (2004) Trigger factor in complex with the

ribosome forms a molecular cradle for nascent proteins. Nature431: 590-596

Flaherty KM, Wilbanks SM, DeLuca-Flaherty C, McKay DB (1994) Structural basis of the 70-kilodalton

heat shock cognate protein ATP hydrolytic activity. II. Structure of the active site with ADP or ATP bound

to wild type and mutant ATPase fragment. J Biol Chem269: 12899-12907

Page 79: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

78

Frydman J, Nimmesgern E, Ohtsuka K, Hartl FU (1994) Folding of nascent polypeptide chains in a high

molecular mass assembly with molecular chaperones. Nature370: 111-117

Fujiwara K, Ishihama Y, Nakahigashi K, Soga T, Taguchi H (2010) A systematic survey of in vivo obligate

chaperonin-dependent substrates. EMBO J29: 1552-1564

Furano AV (1975) Content of elongation factor Tu in Escherichia coli. Proc Natl Acad Sci U S A72: 4780-

4784

Garel JP (1974) Functional adaptation of tRNA population. Journal of theoretical biology43: 211-225

Genest O, Hoskins JR, Camberg JL, Doyle SM, Wickner S (2011) Heat shock protein 90 from Escherichia

coli collaborates with the DnaK chaperone system in client protein remodeling. Proc Natl Acad Sci U S

A108: 8206-8211

Genevaux P, Georgopoulos C, Kelley WL (2007) The Hsp70 chaperone machines of Escherichia coli: a

paradigm for the repartition of chaperone functions. Mol Microbiol66: 840-857

Genevaux P, Keppel F, Schwager F, Langendijk-Genevaux PS, Hartl FU, Georgopoulos C (2004) In vivo

analysis of the overlapping functions of DnaK and trigger factor. EMBO Rep5: 195-200

Genevaux P, Schwager F, Georgopoulos C, Kelley WL (2002) Scanning mutagenesis identifies amino acid

residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain. Genetics162: 1045-

1053

Gerdes K, Christensen SK, Lobner-Olesen A (2005) Prokaryotic toxin-antitoxin stress response loci. Nature

Reviews Microbiology3: 371-382

Gilbert HF (1990) Molecular and cellular aspects of thiol-disulfide exchange. Advances in enzymology and

related areas of molecular biology63: 69-172

Goloubinoff P, Mogk A, Zvi AP, Tomoyasu T, Bukau B (1999) Sequential mechanism of solubilization and

refolding of stable protein aggregates by a bichaperone network. Proc Natl Acad Sci U S A96: 13732-13737

Graf PC, Jakob U (2002) Redox-regulated molecular chaperones. Cell Mol Life Sci59: 1624-1631

Graf PC, Martinez-Yamout M, VanHaerents S, Lilie H, Dyson HJ, Jakob U (2004) Activation of the redox-

regulated chaperone Hsp33 by domain unfolding. J Biol Chem279: 20529-20538

Graubner W, Schierhorn A, Bruser T (2007) DnaK plays a pivotal role in Tat targeting of CueO and

functions beside SlyD as a general Tat signal binding chaperone. J Biol Chem282: 7116-7124

Graumann J, Lilie H, Tang X, Tucker KA, Hoffmann JH, Vijayalakshmi J, Saper M, Bardwell JC, Jakob U

(2001) Activation of the redox-regulated molecular chaperone Hsp33--a two-step mechanism. Structure9:

377-387

Gu SQ, Peske F, Wieden HJ, Rodnina MV, Wintermeyer W (2003) The signal recognition particle binds to

protein L23 at the peptide exit of the Escherichia coli ribosome. Rna9: 566-573

Guenther I, Zolkiewski M, Kedzierska-Mieszkowska S (2012) Cooperation between two ClpB isoforms

enhances the recovery of the recombinant beta-galactosidase from inclusion bodies. Biochem Biophys Res

Commun426: 596-600

Gupta RS (1998) Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships

among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev62: 1435-1491

Page 80: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

79

Guthrie B, Wickner W (1990) Trigger factor depletion or overproduction causes defective cell division but

does not block protein export. J Bacteriol172: 5555-5562

Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level

expression by vectors containing the arabinose PBAD promoter. J Bacteriol177: 4121-4130

Hale CR, Zhao P, Olson S, Duff MO, Graveley BR, Wells L, Terns RM, Terns MP (2009) RNA-guided

RNA cleavage by a CRISPR RNA-Cas protein complex. Cell139: 945-956

Halic M, Gartmann M, Schlenker O, Mielke T, Pool MR, Sinning I, Beckmann R (2006) Signal recognition

particle receptor exposes the ribosomal translocon binding site. Science312: 745-747

Hansen S, Vulic M, Min J, Yen TJ, Schumacher MA, Brennan RG, Lewis K (2012) Regulation of the

Escherichia coli HipBA toxin-antitoxin system by proteolysis. Plos One7: e39185

Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A (2001)

High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell107: 679-688

Harrison CJ, Hayer-Hartl M, Di Liberto M, Hartl F, Kuriyan J (1997) Crystal structure of the nucleotide

exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK. Science276: 431-435

Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature381: 571-579

Hartl FU, Bracher A, Hayer-Hartl M (2011) Molecular chaperones in protein folding and proteostasis.

Nature475: 324-332

Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein.

Science295: 1852-1858

Hartl FU, Hayer-Hartl M (2009) Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol

Biol16: 574-581

Hendrick JP, Hartl FU (1993) Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem62:

349-384

Hesterkamp T, Bukau B (1996) Identification of the prolyl isomerase domain of Escherichia coli trigger

factor. FEBS Lett385: 67-71

Hesterkamp T, Hauser S, Lutcke H, Bukau B (1996) Escherichia coli trigger factor is a prolyl isomerase that

associates with nascent polypeptide chains. Proc Natl Acad Sci U S A93: 4437-4441

Hoffmann A, Becker AH, Zachmann-Brand B, Deuerling E, Bukau B, Kramer G (2012) Concerted action of

the ribosome and the associated chaperone trigger factor confines nascent polypeptide folding. Mol Cell48:

63-74

Hoffmann A, Bukau B, Kramer G (2010) Structure and function of the molecular chaperone Trigger Factor.

Biochimica et Biophysica Acta-Molecular Cell Research1803: 650-661

Hoffmann A, Merz F, Rutkowska A, Zachmann-Brand B, Deuerling E, Bukau B (2006) Trigger factor forms

a protective shield for nascent polypeptides at the ribosome. J Biol Chem281: 6539-6545

Hoffmann JH, Linke K, Graf PC, Lilie H, Jakob U (2004) Identification of a redox-regulated chaperone

network. EMBO J23: 160-168

Page 81: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

80

Hollingsworth MJ, Kim JK, Stollar NE (1998) Heelprinting analysis of in vivo ribosome pause sites.

Methods Mol Biol77: 153-165

Horwich AL, Apetri AC, Fenton WA (2009) The GroEL/GroES cis cavity as a passive anti-aggregation

device. FEBS Lett583: 2654-2662

Houben EN, Zarivach R, Oudega B, Luirink J (2005) Early encounters of a nascent membrane protein:

specificity and timing of contacts inside and outside the ribosome. J Cell Biol170: 27-35

Houry WA, Frishman D, Eckerskorn C, Lottspeich F, Hartl FU (1999) Identification of in vivo substrates of

the chaperonin GroEL. Nature402: 147-154

Huang GC, Li ZY, Zhou JM, Fischer G (2000) Assisted folding of D-glyceraldehyde-3-phosphate

dehydrogenase by trigger factor. Protein Sci9: 1254-1261

Ikemura T (1981) Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence

of the respective codons in its protein genes. J Mol Biol146: 1-21

Ilbert M, Graf PC, Jakob U (2006) Zinc center as redox switch--new function for an old motif. Antioxidants

& redox signaling8: 835-846

Ilbert M, Horst J, Ahrens S, Winter J, Graf PC, Lilie H, Jakob U (2007) The redox-switch domain of Hsp33

functions as dual stress sensor. Nat Struct Mol Biol14: 556-563

Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol57: 395-418

Jackson SE (1998) How do small single-domain proteins fold? Folding & design3: R81-91

Jakob U, Eser M, Bardwell JC (2000) Redox switch of hsp33 has a novel zinc-binding motif. J Biol

Chem275: 38302-38310

Jakob U, Muse W, Eser M, Bardwell JC (1999) Chaperone activity with a redox switch. Cell96: 341-352

Janda I, Devedjiev Y, Derewenda U, Dauter Z, Bielnicki J, Cooper DR, Graf PC, Joachimiak A, Jakob U,

Derewenda ZS (2004) The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33

chaperone and its implications for the activation mechanism. Structure12: 1901-1907

Jong WS, Hagen-Jongman CM, Genevaux P, Brunner J, Oudega B, Luirink J (2004) Trigger factor interacts

with the signal peptide of nascent Tat substrates but does not play a critical role in Tat-mediated export. Eur

J Biochem271: 4779-4787

Kaiser CM, Chang HC, Agashe VR, Lakshmipathy SK, Etchells SA, Hayer-Hartl M, Hartl FU, Barral JM

(2006) Real-time observation of trigger factor function on translating ribosomes. Nature444: 455-460

Kaiser CM, Goldman DH, Chodera JD, Tinoco I, Jr., Bustamante C (2011) The ribosome modulates nascent

protein folding. Science334: 1723-1727

Kandror O, Goldberg AL (1997) Trigger factor is induced upon cold shock and enhances viability of

Escherichia coli at low temperatures. Proc Natl Acad Sci U S A94: 4978-4981

Karzai AW, McMacken R (1996) A bipartite signaling mechanism involved in DnaJ-mediated activation of

the Escherichia coli DnaK protein. J Biol Chem271: 11236-11246

Kedzierska S, Akoev V, Barnett ME, Zolkiewski M (2003) Structure and function of the middle domain of

ClpB from Escherichia coli. Biochemistry42: 14242-14248

Page 82: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

81

Keenan RJ, Freymann DM, Stroud RM, Walter P (2001) The signal recognition particle. Annu Rev

Biochem70: 755-775

Kerner MJ, Naylor DJ, Ishihama Y, Maier T, Chang HC, Stines AP, Georgopoulos C, Frishman D, Hayer-

Hartl M, Mann M, Hartl FU (2005) Proteome-wide analysis of chaperonin-dependent protein folding in

Escherichia coli. Cell122: 209-220

Kim SJ, Jeong DG, Chi SW, Lee JS, Ryu SE (2001) Crystal structure of proteolytic fragments of the redox-

sensitive Hsp33 with constitutive chaperone activity. Nat Struct Biol8: 459-466

Kimsey HH, Dagarag MD, Kumamoto CA (1995) Diverse effects of mutation on the activity of the

Escherichia coli export chaperone SecB. J Biol Chem270: 22831-22835

Kitagawa M, Miyakawa M, Matsumura Y, Tsuchido T (2002) Escherichia coli small heat shock proteins,

IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. Eur J Biochem269: 2907-2917

Kityk R, Kopp J, Sinning I, Mayer MP (2012) Structure and Dynamics of the ATP-Bound Open

Conformation of Hsp70 Chaperones. Mol Cell48: 863-874

Komar AA (2009) A pause for thought along the co-translational folding pathway. Trends Biochem Sci34:

16-24

Komar AA, Lesnik T, Reiss C (1999) Synonymous codon substitutions affect ribosome traffic and protein

folding during in vitro translation. FEBS Lett462: 387-391

Korber P, Stahl JM, Nierhaus KH, Bardwell JC (2000) Hsp15: a ribosome-associated heat shock protein.

EMBO J19: 741-748

Kosolapov A, Deutsch C (2009) Tertiary interactions within the ribosomal exit tunnel. Nat Struct Mol

Biol16: 405-411

Kramer G, Boehringer D, Ban N, Bukau B (2009) The ribosome as a platform for co-translational

processing, folding and targeting of newly synthesized proteins. Nature Structural & Molecular Biology16:

589-597

Kramer G, Patzelt H, Rauch T, Kurz TA, Vorderwulbecke S, Bukau B, Deuerling E (2004a) Trigger factor

peptidyl-prolyl cis/trans isomerase activity is not essential for the folding of cytosolic proteins in Escherichia

coli. J Biol Chem279: 14165-14170

Kramer G, Rauch T, Rist W, Vorderwulbecke S, Patzelt H, Schulze-Specking A, Ban N, Deuerling E, Bukau

B (2002) L23 protein functions as a chaperone docking site on the ribosome. Nature419: 171-174

Kramer G, Rutkowska A, Wegrzyn RD, Patzelt H, Kurz TA, Merz F, Rauch T, Vorderwulbecke S,

Deuerling E, Bukau B (2004b) Functional dissection of Escherichia coli trigger factor: unraveling the

function of individual domains. J Bacteriol186: 3777-3784

Krasheninnikov IA, Komar AA, Adzhubei IA (1989) [Frequency of using codons in mRNA and coding of

the domain structure of proteins]. Doklady Akademii nauk SSSR305: 1006-1012

Krasheninnikov IA, Komar AA, Adzhubei IA (1991) Nonuniform size distribution of nascent globin

peptides, evidence for pause localization sites, and a contranslational protein-folding model. J Protein

Chem10: 445-453

Page 83: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

82

Kristensen O, Gajhede M (2003) Chaperone binding at the ribosomal exit tunnel. Structure (Camb)11: 1547-

1556

Krukenberg KA, Street TO, Lavery LA, Agard DA (2011) Conformational dynamics of the molecular

chaperone Hsp90. Q Rev Biophys44: 229-255

Kuczynska-Wisnik D, Kedzierska S, Matuszewska E, Lund P, Taylor A, Lipinska B, Laskowska E (2002)

The Escherichia coli small heat-shock proteins IbpA and IbpB prevent the aggregation of endogenous

proteins denatured in vivo during extreme heat shock. Microbiology148: 1757-1765

Kudlicki W, Mouat M, Walterscheid JP, Kramer G, Hardesty B (1994) Development of a chaperone-

deficient system by fractionation of a prokaryotic coupled transcription/translation system. Anal

Biochem217: 12-19

Kumamoto CA, Beckwith J (1985) Evidence for specificity at an early step in protein export in Escherichia

coli. J Bacteriol163: 267-274

Kumar M, Sourjik V (2012) Physical map and dynamics of the chaperone network in Escherichia coli. Mol

Microbiol84: 736-747

Kumsta C, Jakob U (2009) Redox-regulated chaperones. Biochemistry48: 4666-4676

Lakshmipathy SK, Gupta R, Pinkert S, Etchells SA, Hartl FU (2010) Versatility of trigger factor interactions

with ribosome-nascent chain complexes. J Biol Chem285: 27911-27923

Lakshmipathy SK, Tomic S, Kaiser CM, Chang HC, Genevaux P, Georgopoulos C, Barral JM, Johnson AE,

Hartl FU, Etchells SA (2007) Identification of nascent chain interaction sites on trigger factor. J Biol

Chem282: 12186-12193

Langer T, Lu C, Echols H, Flanagan J, Hayer MK, Hartl FU (1992) Successive action of DnaK, DnaJ and

GroEL along the pathway of chaperone-mediated protein folding. Nature356: 683-689

Laskowska E, Wawrzynow A, Taylor A (1996) IbpA and IbpB, the new heat-shock proteins, bind to

endogenous Escherichia coli proteins aggregated intracellularly by heat shock. Biochimie78: 117-122

Laufen T, Mayer MP, Beisel C, Klostermeier D, Mogk A, Reinstein J, Bukau B (1999) Mechanism of

regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A96: 5452-5457

Lecker S, Lill R, Ziegelhoffer T, Georgopoulos C, Bassford PJ, Jr., Kumamoto CA, Wickner W (1989)

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes

with precursor proteins in vitro. EMBO J8: 2703-2709

Lee HC, Bernstein HD (2002) Trigger factor retards protein export in Escherichia coli. J Biol Chem277:

43527-43535

Lee S, Sowa ME, Watanabe YH, Sigler PB, Chiu W, Yoshida M, Tsai FT (2003) The structure of ClpB: a

molecular chaperone that rescues proteins from an aggregated state. Cell115: 229-240

Leichert LI, Gehrke F, Gudiseva HV, Blackwell T, Ilbert M, Walker AK, Strahler JR, Andrews PC, Jakob U

(2008) Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. Proc Natl Acad Sci U

S A105: 8197-8202

Lewis K (2010) Persister cells. Annu Rev Microbiol64: 357-372

Page 84: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

83

Li H, Chang L, Howell JM, Turner RJ (2010) DmsD, a Tat system specific chaperone, interacts with other

general chaperones and proteins involved in the molybdenum cofactor biosynthesis. Biochim Biophys

Acta1804: 1301-1309

Liberek K, Marszalek J, Ang D, Georgopoulos C, Zylicz M (1991) Escherichia coli DnaJ and GrpE heat

shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A88: 2874-2878

Lin KF, Sun CS, Huang YC, Chan SI, Koubek J, Wu TH, Huang JJ (2012) Cotranslational protein folding

within the ribosome tunnel influences trigger-factor recruitment. Biophys J102: 2818-2827

Lin Z, Madan D, Rye HS (2008) GroEL stimulates protein folding through forced unfolding. Nat Struct Mol

Biol15: 303-311

Lu J, Deutsch C (2005) Folding zones inside the ribosomal exit tunnel. Nat Struct Mol Biol12: 1123-1129

Lu J, Kobertz WR, Deutsch C (2007) Mapping the electrostatic potential within the ribosomal exit tunnel. J

Mol Biol371: 1378-1391

Luirink J, High S, Wood H, Giner A, Tollervey D, Dobberstein B (1992) Signal-sequence recognition by an

Escherichia coli ribonucleoprotein complex. Nature359: 741-743

Lukash TO, Turkivska HV, Negrutskii BS, El'skaya AV (2004) Chaperone-like activity of mammalian

elongation factor eEF1A: renaturation of aminoacyl-tRNA synthetases. Int J Biochem Cell Biol36: 1341-

1347

Lyon WR, Caparon MG (2003) Trigger factor-mediated prolyl isomerization influences maturation of the

Streptococcus pyogenes cysteine protease. J Bacteriol185: 3661-3667

Maillard J, Genevaux P, Holliger C (2011) Redundancy and specificity of multiple trigger factor chaperones

in Desulfitobacteria. Microbiology157: 2410-2421

Maki JA, Schnobrich DJ, Culver GM (2002) The DnaK chaperone system facilitates 30S ribosomal subunit

assembly. Mol Cell10: 129-138

Malkin LI, Rich A (1967) Partial resistance of nascent polypeptide chains to proteolytic digestion due to

ribosomal shielding. J Mol Biol26: 329-346

Marin M (2008) Folding at the rhythm of the rare codon beat. Biotechnology journal3: 1047-1057

Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontal gene transfer in staphylococci

by targeting DNA. Science322: 1843-1845

Martinez-Hackert E, Hendrickson WA (2007) Structures of and interactions between domains of trigger

factor from Thermotoga maritima. Acta Crystallogr D Biol Crystallogr63: 536-547

Martinez-Hackert E, Hendrickson WA (2009) Promiscuous substrate recognition in folding and assembly

activities of the trigger factor chaperone. Cell138: 923-934

Matuszewska E, Kwiatkowska J, Kuczynska-Wisnik D, Laskowska E (2008) Escherichia coli heat-shock

proteins IbpA/B are involved in resistance to oxidative stress induced by copper. Microbiology154: 1739-

1747

Maurizi MR, Clark WP, Katayama Y, Rudikoff S, Pumphrey J, Bowers B, Gottesman S (1990) Sequence

and structure of Clp P, the proteolytic component of the ATP-dependent Clp protease of Escherichia coli. J

Biol Chem265: 12536-12545

Page 85: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

84

Mayer MP (2010) Gymnastics of molecular chaperones. Mol Cell39: 321-331

Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life

Sci62: 670-684

Mayhew M, da Silva AC, Martin J, Erdjument-Bromage H, Tempst P, Hartl FU (1996) Protein folding in the

central cavity of the GroEL-GroES chaperonin complex. Nature379: 420-426

Meriin AB, Mense M, Colbert JD, Liang F, Bihler H, Zaarur N, Rock KL, Sherman MY (2012) A novel

approach to recovery of function of mutant proteins by slowing down translation. J Biol Chem287: 34264-

34272

Merz F, Boehringer D, Schaffitzel C, Preissler S, Hoffmann A, Maier T, Rutkowska A, Lozza J, Ban N,

Bukau B, Deuerling E (2008) Molecular mechanism and structure of Trigger Factor bound to the translating

ribosome. EMBO J27: 1622-1632

Merz F, Hoffmann A, Rutkowska A, Zachmann-Brand B, Bukau B, Deuerling E (2006) The C-terminal

domain of Escherichia coli trigger factor represents the central module of its chaperone activity. J Biol

Chem281: 31963-31971

Mogk A, Deuerling E, Vorderwulbecke S, Vierling E, Bukau B (2003a) Small heat shock proteins, ClpB and

the DnaK system form a functional triade in reversing protein aggregation. Mol Microbiol50: 585-595

Mogk A, Schlieker C, Strub C, Rist W, Weibezahn J, Bukau B (2003b) Roles of individual domains and

conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity.

J Biol Chem278: 17615-17624

Mogk A, Tomoyasu T, Goloubinoff P, Rudiger S, Roder D, Langen H, Bukau B (1999) Identification of

thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB. EMBO J

18: 6934-6949

Moll I, Engelberg-Kulka H (2012) Selective translation during stress in Escherichia coli. Trends Biochem

Sci37: 493-498

Motohashi K, Watanabe Y, Yohda M, Yoshida M (1999) Heat-inactivated proteins are rescued by the

DnaK.J-GrpE set and ClpB chaperones. Proc Natl Acad Sci U S A96: 7184-7189

Motojima F, Motojima-Miyazaki Y, Yoshida M (2012) Revisiting the contribution of negative charges on

the chaperonin cage wall to the acceleration of protein folding. Proc Natl Acad Sci U S A109: 15740-15745

Muller JP (1996) Influence of impaired chaperone or secretion function on SecB production in Escherichia

coli. J Bacteriol178: 6097-6104

Nagy M, Guenther I, Akoyev V, Barnett ME, Zavodszky MI, Kedzierska-Mieszkowska S, Zolkiewski M

(2010) Synergistic cooperation between two ClpB isoforms in aggregate reactivation. J Mol Biol396: 697-

707

Nissen P, Hansen J, Ban N, Moore PB, Steitz TA (2000) The structural basis of ribosome activity in peptide

bond synthesis. Science289: 920-930

Niwa T, Kanamori T, Ueda T, Taguchi H (2012) Global analysis of chaperone effects using a reconstituted

cell-free translation system. Proc Natl Acad Sci U S A109: 8937-8942

Page 86: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

85

O'Brien EP, Christodoulou J, Vendruscolo M, Dobson CM (2012) Trigger factor slows co-translational

folding through kinetic trapping while sterically protecting the nascent chain from aberrant cytosolic

interactions. J Am Chem Soc134: 10920-10932

Oh E, Becker AH, Sandikci A, Huber D, Chaba R, Gloge F, Nichols RJ, Typas A, Gross CA, Kramer G,

Weissman JS, Bukau B (2011) Selective ribosome profiling reveals the cotranslational chaperone action of

trigger factor in vivo. Cell147: 1295-1308

Onuchic JN, Wolynes PG (2004) Theory of protein folding. Curr Opin Struct Biol14: 70-75

Oresnik IJ, Ladner CL, Turner RJ (2001) Identification of a twin-arginine leader-binding protein. Mol

Microbiol40: 323-331

Osorio H, Carvalho E, del Valle M, Gunther Sillero MA, Moradas-Ferreira P, Sillero A (2003) H2O2, but

not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces

cerevisiae. An experimental and theoretical approach. Eur J Biochem270: 1578-1589

Pal D, Chattopadhyay S, Chandra S, Sarkar D, Chakraborty A, Das Gupta C (1997) Reactivation of

denatured proteins by domain V of bacterial 23S rRNA. Nucleic Acids Res25: 5047-5051

Patzelt H, Rudiger S, Brehmer D, Kramer G, Vorderwulbecke S, Schaffitzel E, Waitz A, Hesterkamp T,

Dong L, Schneider-Mergener J, Bukau B, Deuerling E (2001) Binding specificity of Escherichia coli trigger

factor. Proc Natl Acad Sci U S A98: 14244-14249

Pearl LH, Prodromou C (2006) Structure and mechanism of the Hsp90 molecular chaperone machinery.

Annu Rev Biochem75: 271-294

Perez-Rodriguez R, Fisher AC, Perlmutter JD, Hicks MG, Chanal A, Santini CL, Wu LF, Palmer T, DeLisa

MP (2007) An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate

proteins of the bacterial twin-arginine translocation pathway. J Mol Biol367: 715-730

Phillips GJ, Silhavy TJ (1990) Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid

proteins in E. coli. Nature344: 882-884

Picking WD, Picking WL, Odom OW, Hardesty B (1992) Fluorescence characterization of the environment

encountered by nascent polyalanine and polyserine as they exit Escherichia coli ribosomes during

translation. Biochemistry31: 2368-2375

Preissler S, Deuerling E (2012) Ribosome-associated chaperones as key players in proteostasis. Trends

Biochem Sci37: 274-283

Purvis IJ, Bettany AJ, Santiago TC, Coggins JR, Duncan K, Eason R, Brown AJ (1987) The efficiency of

folding of some proteins is increased by controlled rates of translation in vivo. A hypothesis. J Mol Biol193:

413-417

Qi HY, Hyndman JB, Bernstein HD (2002) DnaK promotes the selective export of outer membrane protein

precursors in SecA-deficient Escherichia coli. J Biol Chem277: 51077-51083

Radford SE, Dobson CM, Evans PA (1992) The folding of hen lysozyme involves partially structured

intermediates and multiple pathways. Nature358: 302-307

Raine A, Lovmar M, Wikberg J, Ehrenberg M (2006) Trigger factor binding to ribosomes with nascent

peptide chains of varying lengths and sequences. J Biol Chem281: 28033-28038

Page 87: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

86

Raman B, Siva Kumar LV, Ramakrishna T, Mohan Rao C (2001) Redox-regulated chaperone function and

conformational changes of Escherichia coli Hsp33. FEBS Lett489: 19-24

Randall LL, Hardy SJ (2002) SecB, one small chaperone in the complex milieu of the cell. Cell Mol Life

Sci59: 1617-1623

Ratajczak E, Zietkiewicz S, Liberek K (2009) Distinct activities of Escherichia coli small heat shock proteins

IbpA and IbpB promote efficient protein disaggregation. J Mol Biol386: 178-189

Reichmann D, Xu Y, Cremers CM, Ilbert M, Mittelman R, Fitzgerald MC, Jakob U (2012) Order out of

disorder: working cycle of an intrinsically unfolded chaperone. Cell148: 947-957

Rodrigue A, Batia N, Muller M, Fayet O, Bohm R, Mandrand-Berthelot MA, Wu LF (1996) Involvement of

the GroE chaperonins in the nickel-dependent anaerobic biosynthesis of NiFe-hydrogenases of Escherichia

coli. J Bacteriol178: 4453-4460

Rojiani MV, Jakubowski H, Goldman E (1990) Relationship between protein synthesis and concentrations of

charged and uncharged tRNATrp in Escherichia coli. Proc Natl Acad Sci U S A87: 1511-1515

Rousseau F, Serrano L, Schymkowitz JW (2006) How evolutionary pressure against protein aggregation

shaped chaperone specificity. J Mol Biol355: 1037-1047

Rudiger S, Germeroth L, Schneider-Mergener J, Bukau B (1997) Substrate specificity of the DnaK

chaperone determined by screening cellulose-bound peptide libraries. EMBO J16: 1501-1507

Rutkowska A, Mayer MP, Hoffmann A, Merz F, Zachmann-Brand B, Schaffitzel C, Ban N, Deuerling E,

Bukau B (2008) Dynamics of trigger factor interaction with translating ribosomes. J Biol Chem 283: 4124-

4132

Sakr S, Cirinesi AM, Ullers RS, Schwager F, Georgopoulos C, Genevaux P (2010) Lon Protease Quality

Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40)

Chaperones. J Biol Chem285: 23504-23512

Sargent F (2007) The twin-arginine transport system: moving folded proteins across membranes. Biochem

Soc Trans35: 835-847

Sargent F, Bogsch EG, Stanley NR, Wexler M, Robinson C, Berks BC, Palmer T (1998) Overlapping

functions of components of a bacterial Sec-independent protein export pathway. EMBO J17: 3640-3650

Sauer RT, Baker TA (2011) AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev

Biochem80: 587-612

Schiebel E, Driessen AJ, Hartl FU, Wickner W (1991) Delta mu H+ and ATP function at different steps of

the catalytic cycle of preprotein translocase. Cell64: 927-939

Schirmer EC, Glover JR, Singer MA, Lindquist S (1996) HSP100/Clp proteins: a common mechanism

explains diverse functions. Trends Biochem Sci21: 289-296

Schlecht R, Erbse AH, Bukau B, Mayer MP (2011) Mechanics of Hsp70 chaperones enables differential

interaction with client proteins. Nat Struct Mol Biol18: 345-351

Schlunzen F, Wilson DN, Tian P, Harms JM, McInnes SJ, Hansen HA, Albrecht R, Buerger J, Wilbanks

SM, Fucini P (2005) The binding mode of the trigger factor on the ribosome: implications for protein folding

and SRP interaction. Structure (Camb)13: 1685-1694

Page 88: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

87

Scholz C, Stoller G, Zarnt T, Fischer G, Schmid FX (1997) Cooperation of enzymatic and chaperone

functions of trigger factor in the catalysis of protein folding. EMBO J16: 54-58

Schroder H, Langer T, Hartl FU, Bukau B (1993) DnaK, DnaJ and GrpE form a cellular chaperone

machinery capable of repairing heat-induced protein damage. EMBO J12: 4137-4144

Schumacher MA, Piro KM, Xu W, Hansen S, Lewis K, Brennan RG (2009) Molecular mechanisms of

HipA-mediated multidrug tolerance and its neutralization by HipB. Science323: 396-401

Seyffer F, Kummer E, Oguchi Y, Winkler J, Kumar M, Zahn R, Sourjik V, Bukau B, Mogk A (2012) Hsp70

proteins bind Hsp100 regulatory M domains to activate AAA+ disaggregase at aggregate surfaces. Nat Struct

Mol Biol19: 1347-1355

Sharma SK, De los Rios P, Christen P, Lustig A, Goloubinoff P (2010) The kinetic parameters and energy

cost of the Hsp70 chaperone as a polypeptide unfoldase. Nature chemical biology6: 914-920

Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, Wright F (1988) Codon usage patterns in

Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila

melanogaster and Homo sapiens; a review of the considerable within-species diversity. Nucleic Acids Res16:

8207-8211

Shearstone JR, Baneyx F (1999) Biochemical characterization of the small heat shock protein IbpB from

Escherichia coli. J Biol Chem274: 9937-9945

Shiina N, Gotoh Y, Kubomura N, Iwamatsu A, Nishida E (1994) Microtubule severing by elongation factor

1 alpha. Science266: 282-285

Siegers K, Waldmann T, Leroux MR, Grein K, Shevchenko A, Schiebel E, Hartl FU (1999)

Compartmentation of protein folding in vivo: sequestration of non-native polypeptide by the chaperonin-

GimC system. EMBO J18: 75-84

Siller E, DeZwaan DC, Anderson JF, Freeman BC, Barral JM (2010) Slowing bacterial translation speed

enhances eukaryotic protein folding efficiency. J Mol Biol396: 1310-1318

Spence J, Cegielska A, Georgopoulos C (1990) Role of Escherichia coli heat shock proteins DnaK and HtpG

(C62.5) in response to nutritional deprivation. J Bacteriol172: 7157-7166

Squires C, Squires CL (1992) The Clp proteins: proteolysis regulators or molecular chaperones? J

Bacteriol174: 1081-1085

Squires CL, Pedersen S, Ross BM, Squires C (1991) ClpB is the Escherichia coli heat shock protein F84.1. J

Bacteriol173: 4254-4262

Stoller G, Rucknagel KP, Nierhaus KH, Schmid FX, Fischer G, Rahfeld JU (1995) A ribosome-associated

peptidyl-prolyl cis/trans isomerase identified as the trigger factor. EMBO J14: 4939-4948

Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Microbiol2: 188-194

Straus D, Walter W, Gross CA (1990) DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat

shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev4: 2202-2209

Swain JF, Dinler G, Sivendran R, Montgomery DL, Stotz M, Gierasch LM (2007) Hsp70 chaperone ligands

control domain association via an allosteric mechanism mediated by the interdomain linker. Mol Cell26: 27-

39

Page 89: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

88

Tang YC, Chang HC, Chakraborty K, Hartl FU, Hayer-Hartl M (2008) Essential role of the chaperonin

folding compartment in vivo. EMBO J27: 1458-1468

Tang YC, Chang HC, Roeben A, Wischnewski D, Wischnewski N, Kerner MJ, Hartl FU, Hayer-Hartl M

(2006) Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein.

Cell125: 903-914

Tartaglia GG, Dobson CM, Hartl FU, Vendruscolo M (2010) Physicochemical determinants of chaperone

requirements. J Mol Biol400: 579-588

Tartaglia GG, Pechmann S, Dobson CM, Vendruscolo M (2007) Life on the edge: a link between gene

expression levels and aggregation rates of human proteins. Trends Biochem Sci32: 204-206

Teter SA, Houry WA, Ang D, Tradler T, Rockabrand D, Fischer G, Blum P, Georgopoulos C, Hartl FU

(1999) Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning

nascent chains. Cell97: 755-765

Thanaraj TA, Argos P (1996) Protein secondary structural types are differentially coded on messenger RNA.

Protein Sci5: 1973-1983

Thomas JG, Baneyx F (1998) Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in

thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo. J Bacteriol180: 5165-5172

Thomas PJ, Qu BH, Pedersen PL (1995) Defective protein folding as a basis of human disease. Trends

Biochem Sci20: 456-459

Thompson RC, Dix DB, Karim AM (1986) The reaction of ribosomes with elongation factor Tu.GTP

complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of

protein synthesis. J Biol Chem261: 4868-4874

Tomoyasu T, Arsene F, Ogura T, Bukau B (2001a) The C terminus of sigma(32) is not essential for

degradation by FtsH. JBacteriol183: 5911-5917

Tomoyasu T, Mogk A, Langen H, Goloubinoff P, Bukau B (2001b) Genetic dissection of the roles of

chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol. Mol

Microbiol40: 397-413

Travers AA, Kamen RI, Schleif RF (1970) Factor necessary for ribosomal RNA synthesis. Nature228: 748-

751

Turner RJ, Papish AL, Sargent F (2004) Sequence analysis of bacterial redox enzyme maturation proteins

(REMPs). Can J Microbiol50: 225-238

Ullers RS, Ang D, Schwager F, Georgopoulos C, Genevaux P (2007) Trigger Factor can antagonize both

SecB and DnaK/DnaJ chaperone functions in Escherichia coli. Proc Natl Acad Sci U S A104: 3101-3106

Ullers RS, Houben EN, Raine A, ten Hagen-Jongman CM, Ehrenberg M, Brunner J, Oudega B, Harms N,

Luirink J (2003) Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit

site on the Escherichia coli ribosome. J Cell Biol161: 679-684

Ullers RS, Luirink J, Harms N, Schwager F, Georgopoulos C, Genevaux P (2004) SecB is a bona fide

generalized chaperone in Escherichia coli. Proc Natl Acad Sci U S A101: 7583-7588

Page 90: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

89

Urbanus ML, Scotti PA, Froderberg L, Saaf A, de Gier JW, Brunner J, Samuelson JC, Dalbey RE, Oudega

B, Luirink J (2001) Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with

SecY and YidC. EMBO Rep2: 524-529

Valent QA, Kendall DA, High S, Kusters R, Oudega B, Luirink J (1995) Early events in preprotein

recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides. EMBO J14: 5494-

5505

Van den Berg B, Clemons WM, Jr., Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA (2004)

X-ray structure of a protein-conducting channel. Nature427: 36-44

Van Melderen L, De Bast MS (2009) Bacterial Toxin-Antitoxin Systems: More Than Selfish Entities? Plos

Genetics5

Vijayalakshmi J, Mukhergee MK, Graumann J, Jakob U, Saper MA (2001) The 2.2 A crystal structure of

Hsp33: a heat shock protein with redox-regulated chaperone activity. Structure9: 367-375

Vogel M, Bukau B, Mayer MP (2006a) Allosteric regulation of Hsp70 chaperones by a proline switch. Mol

Cell21: 359-367

Vogel M, Mayer MP, Bukau B (2006b) Allosteric regulation of Hsp70 chaperones involves a conserved

interdomain linker. J Biol Chem281: 38705-38711

Voisset C, Thuret JY, Tribouillard-Tanvier D, Saupe SJ, Blondel M (2008) Tools for the study of ribosome-

borne protein folding activity. Biotechnology journal3: 1033-1040

Vorderwulbecke S, Kramer G, Merz F, Kurz TA, Rauch T, Zachmann-Brand B, Bukau B, Deuerling E

(2004) Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking

trigger factor and DnaK. FEBS Lett559: 181-187

Voss NR, Gerstein M, Steitz TA, Moore PB (2006) The geometry of the ribosomal polypeptide exit tunnel. J

Mol Biol360: 893-906

Wandinger SK, Richter K, Buchner J (2008) The Hsp90 chaperone machinery. J Biol Chem283: 18473-

18477

Ward JE, Jr., Lutkenhaus J (1985) Overproduction of FtsZ induces minicell formation in E. coli. Cell42:

941-949

Weibezahn J, Tessarz P, Schlieker C, Zahn R, Maglica Z, Lee S, Zentgraf H, Weber-Ban EU, Dougan DA,

Tsai FT, Mogk A, Bukau B (2004) Thermotolerance requires refolding of aggregated proteins by substrate

translocation through the central pore of ClpB. Cell119: 653-665

Weissman JS, Rye HS, Fenton WA, Beechem JM, Horwich AL (1996) Characterization of the active

intermediate of a GroEL-GroES-mediated protein folding reaction. Cell84: 481-490

Wholey WY, Jakob U (2012) Hsp33 confers bleach resistance by protecting elongation factor Tu against

oxidative degradation in Vibrio cholerae. Mol Microbiol83: 981-991

Widmann M, Clairo M, Dippon J, Pleiss J (2008) Analysis of the distribution of functionally relevant rare

codons. BMC genomics9: 207

Wild J, Altman E, Yura T, Gross CA (1992) DnaK and DnaJ heat shock proteins participate in protein export

in Escherichia coli. Genes Dev6: 1165-1172

Page 91: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

90

Wild J, Rossmeissl P, Walter WA, Gross CA (1996) Involvement of the DnaK-DnaJ-GrpE chaperone team

in protein secretion in Escherichia coli. J Bacteriol178: 3608-3613

Winkler J, Seybert A, Konig L, Pruggnaller S, Haselmann U, Sourjik V, Weiss M, Frangakis AS, Mogk A,

Bukau B (2010) Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and

consequences on protein quality control and cellular ageing. EMBO J 29: 910-923

Winkler J, Tyedmers J, Bukau B, Mogk A (2012) Hsp70 targets Hsp100 chaperones to substrates for protein

disaggregation and prion fragmentation. J Cell Biol198: 387-404

Winter J, Ilbert M, Graf PC, Ozcelik D, Jakob U (2008) Bleach activates a redox-regulated chaperone by

oxidative protein unfolding. Cell135: 691-701

Winter J, Linke K, Jatzek A, Jakob U (2005) Severe oxidative stress causes inactivation of DnaK and

activation of the redox-regulated chaperone Hsp33. Mol Cell17: 381-392

Wolin SL, Walter P (1988) Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO

J7: 3559-3569

Won HS, Low LY, Guzman RD, Martinez-Yamout M, Jakob U, Dyson HJ (2004) The zinc-dependent redox

switch domain of the chaperone Hsp33 has a novel fold. J Mol Biol341: 893-899

Woolhead CA, McCormick PJ, Johnson AE (2004) Nascent membrane and secretory proteins differ in

FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell116: 725-736

Xu Z, Horwich AL, Sigler PB (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7

chaperonin complex. Nature388: 741-750

Yamaguchi Y, Park JH, Inouye M (2011) Toxin-antitoxin systems in bacteria and archaea. Annual review of

genetics45: 61-79

Yamaguchi Y, Tomoyasu T, Takaya A, Morioka M, Yamamoto T (2003) Effects of disruption of heat shock

genes on susceptibility of Escherichia coli to fluoroquinolones. BMC Microbiol3: 16

Yang F, Demma M, Warren V, Dharmawardhane S, Condeelis J (1990) Identification of an actin-binding

protein from Dictyostelium as elongation factor 1a. Nature347: 494-496

Yosef I, Goren MG, Kiro R, Edgar R, Qimron U (2011) High-temperature protein G is essential for activity

of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system. Proc

Natl Acad Sci U S A108: 20136-20141

Young JC, Agashe VR, Siegers K, Hartl FU (2004) Pathways of chaperone-mediated protein folding in the

cytosol. Nat Rev Mol Cell Biol5: 781-791

Zarnt T, Tradler T, Stoller G, Scholz C, Schmid FX, Fischer G (1997) Modular structure of the trigger factor

required for high activity in protein folding. J Mol Biol271: 827-837

Zhang T, Ploetz EA, Nagy M, Doyle SM, Wickner S, Smith PE, Zolkiewski M (2012) Flexible connection of

the N-terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency.

Proteins80: 2758-2768

Zheng M, Aslund F, Storz G (1998) Activation of the OxyR transcription factor by reversible disulfide bond

formation. Science279: 1718-1721

Page 92: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

91

Zolkiewski M (1999) ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A

novel multi-chaperone system from Escherichia coli. J Biol Chem274: 28083-28086

Page 93: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

92

Page 94: T THHÈÈSSEE - thesesupsthesesups.ups-tlse.fr/2098/1/2013TOU30049.pdfhuman diseases (Fig. 1) (Barral et al, 2004; Dobson, 2001; Thomas et al, 1995). To counteract To counteract such

93

REMERCIEMENTS

Je remercie sincèrement les membres du jury d'être venu d'aussi loin afin d’évaluer ce travail.

Je remercie mon directeur de thèse, Pierre ainsi que Marie-Pierre (co-directeur) et Joen (co-

superviseur), pour leurs conseils avisés et leur aide inestimable tout au long de ces années. J’ai

appris énormément de choses à la fois au niveau scientifique et logistique grâce à eux.

Je remercie tous les membres de l’équipe Genevaux, passés et présents: Patricia, Anne-Marie, Elsa,

Ambre, Frédéric, et Sam ainsi que mes deux stagiaires Gaëlle et Thomas. J’y ai rencontré des

personnes formidables qui m’ont apporté un réel soutien scientifique et humain et qui sont devenus

pour moi de très bons amis.

Je remercie les membres de l’équipe de Joen Luirink de l’Université Libre d’Amsterdam: Joen,

Ana, Zora, Sadeeq, Wouter et Gregory de m’avoir accueilli pendant plus de quatre mois afin

d’apprendre et d’appliquer une expérience de traduction in vitro dans des conditions optimales et

pour leur bonne humeur et leur soutien sans failles.

Merci à mes collègues du LMGM avec qui j'ai passé d'excellents moments: Laure, Cécile, Anne,

Brigitte, Véronique en particulier.

Mes travaux ont été financés par une bourse CNRS/Région Midi-Pyrénées. Je remercie donc le

CNRS ainsi que la région Midi-Pyrénées pour leur soutien financier.

Merci à mes parents et à ma petite sœur sans qui je n’aurais jamais pu aller aussi loin dans la vie,

sachant que la route n’est pas encore terminée. Merci de m’avoir laissé accomplir mes rêves.

Merci à Ludo, mon coloc préféré, qui a fait de cette thèse une expérience unique. Nos soirées à

deux vont me manquer.

Et merci à Alexane, un papillon qui s’est posé sur la route de ma vie.