Identification and characterization of novel regulators in …aok.pte.hu/docs/phd/file/dolgozatok/2010/Hocsak_Eniko_PhD_dolgozat.pdf · Identification and characterization of novel

Identification and characterization of novel regulators in

mitochondria related cell death

Ph.D. Thesis

Enikő Hocsák

Program leader: Professor Balázs Sümegi, DSci

University of Pécs, Medical School

Department of Biochemistry and Medical Chemistry

Hungary

2010

Hocsák, E., Identification and characterization of novel regulators

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1.Contents

1.Contents 2

2. Abbreviations 4

3. Introduction 6

3.1. The role of mitochondria in cell death 6

3.1.1. Mitocondrial Membrane Polarization (∆ψ) 7

3.1.2. The mitochondrial permeability transition (MPT) 7

3.1.3. BH3 domain proteins, the Bcl-2 family 9

3.2. The TIP47 protein 11

3.3. SOUL/HBP2, a novel BH3 domain protein 13

4. Aims of the study 15

5. Materials and methods 16

5.1. Chemicals and antibodies 16

5.2. Preparation of polyclonal antibodies 16

5.3. Construction of expression plasmids, expression and purification 17

5.4. Stable transfection 17

5.5. Cell culture and sample preparation 17

5.6. Western blot analysis 18

5.7. Suppression of TIP47 expression by diced siRNA technique 18

5.8. Cell viability assay 19

5.9. Determination of intracellular reactive oxygen species (ROS) 19

5.10. Detecting mitochondrial membrane potential (∆ψ) 19

5.11. Isolation of mitochondria for the in vitro study 20

5.12. Annexin V-FITC assay 20

5.13. Flow cytometry analysis 21

5.14. Statistical analysis 21

6. Results 22

6.1. TIP47 protects mitochondrial membrane integrity and inhibits cell death 22

6.1.1. Effect of expression of TIP47 on H2O2 or taxol-induced cell death 22

6.1.2. The effect of taxol treatment on the cell viability of HeLa cells

transfected with TIP47 siRNA 23

6.1.3. Induction of apoptosis by H2O2, Taxol and etoposide in NIH3T3 cells 24


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6.1.4. Detection of H2O2 induced apoptosis by flow cytometry 26

6.1.5. Localization of TIP47 in oxidative stress 27

6.1.6. Effect of recombinant TIP47 on the mPT and ∆ψ in Vitro 27

6.1.7. The effect of TIP47 on release of mitochondrial intermembrane

space proteins from living cells 30

6.1.8. Effect of TIP47 on the mitochondrial membrane potential in NIH3T3

cells 31

6.1.9. Effect of TIP47 on ROS-production 32

6.1.10. The effect of TIP47 on the pro- and anti-apoptotic signaling

pathways 33

6.2. Cell death in oxidative stress by a novel BH3 domain protein 35

6.2.1. Characterization of SOUL protein 35

6.2.2.Determination of the role of mPT complex in the SOUL-induced

sensitization toward oxidative stress 35

6.2.3. Effect of deletion of the putative BH3 domain on the cell death

facilitating effect of SOUL 37

6.2.4. Effect of SOUL on cell death is inhibited by the overexpression of

anti-apoptotic proteins 38

6.2.5. Effect of SOUL on the hydrogen-peroxide-induced collapse of MMP 42

7. Discussion 44

8. Conclusions 49

9. Acknowledgements 52

10. References 53

11. List of Publications 63


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2. Abbreviations

A23187 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-

methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec2-yl}methyl)-

1,3-benzoxazole-4- carboxylic acid

ADRP adipose differentiation-related protein

AIF apoptosis inducing factor

ANT adenine nucleotide translocase

Bcl-2 B-cell lymphoma

C-400 5-(and-6)-carboxy-2', 7’-dichlorodihydrofluorescein diacetate

CIN cervical intraepithelial neoplasia

CypD cyclophilin D

cyt-c cytochrome c

CsA, cyclosporine A

DIABLO direct IAP–binding protein of low isoelectric point [pI]

DMEM Dulbecco’s modified Eagle’s medium

dsiRNA diced small interfering ribonucleic acid

Endo G endonuclease G

Etoposide 4' - demethyl - epipodophyllotoxin 9 - [4,6-O-(R)-ethylidene-beta-D-

glucopyranoside], 4' - (dihydrogen phosphate)

FCS fetal calf serum

FITC fluorescein isothiocyanate

G418 (2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-

[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-

hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol

GFP green fluorescent protein

GST glutathione S-transferase

H2O2 hydrogen-peroxide

HBP22 heme binding protein 1

HIV-1 Human Immunodeficiency Virus

IAP inhibitor of apoptosis binding protein

IPTG isopropyl-β-D-thiogalactopyranoside

JC-1 5,5,6,6-tetracloro-1,1,3,3 -tetraethylbenzimidazolyl-carbocyanine iodide


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M6PR mannose 6-phosphate receptors

MMP mitochondrial membrane permeabilization

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

mPT mitochondrial permeability transition

∆ψ mitochondrial membrane potential

NCBI National Center for Biotechnology Information

PAT perilipin, adipophilin, and TIP47

PBS phosphate buffered saline

PCR Polymerase Chain Reaction

pI isoelectric point

PI propidium iodide

PP17b placental tissue protein 17b

PTP permeability transition pore

PTPC permeability transition pore complex

Rh-123 rhodamine-1,2,3

ROS reactive oxygen species

siRNA small interfering RNA

Smac second mitochondrial activator of caspases

SOUL/HBP2 heme binding protein 2

Taxol (2α,4α,5β,7β,10β,13α)-4,10-bis (acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-

hydroxy-3-phenylpropanoyl]oxy} -1,7-dihydroxy-9-oxo-5,20-epoxytax-11-

en-2-yl benzoate

TIP47 tail interacting protein of 47 kDa

t-TIP47 truncated-TIP47

VDAC voltage-dependent anionic channel


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3. Introduction

For the past 15 years our laboratory has been involved of identified several genes with

previously unknown functions that affected cell death processes such as tail interacting protein

of 47 kDa (TIP47) or Heme Binding Protein 2 (HBP2/ SOUL). In this study the effect of these

proteins in mitochondrial releated cell death is described.

This work provides compelling evidence of the protective effect of TIP47 which exerts an

antiapoptotic effect on cell survival by inhibiting the activation of proapoptotic proteins, by

assisting the effect of anti-apoptotic proteins and by the direct stabilization of the

mitochondrial membrane potential.

We identified genes, which in contrast, destabilize the mitochondrial membrane system,

such as SOUL, which sensitized fibroblast cells to cell death in the absence of elevated ROS

formation. In the presence of subthreshold calcium, recombinant SOUL provoked

mitochondrial permeability transition (mPT) in vitro, which was inhibited by cyclosporine A

(CsA). In vivo, SOUL overexpression enhanced the dissipation of mitochondrial membrane

potential, as well as SOUL promoted necrotic death in etoposide and A23187 treated cells,

which effect was prevented by CsA.

This Thesis presents my own individual research efforts with my colleague team members.

3.1. The role of mitochondria in cell death

The cell’s decision to die from necrosis or apoptosis is dictated at least in part by the

abundance of intracellular energy stores. Indeed, whereas apoptosis requires a minimal amount

of intracellular ATP, necrosis is generally accompanied by its total depletion [1-2].

The intrinsic cell death pathway involves the initiation of apoptosis as a result of a

disturbance of intracellular homeostasis. In this pathway, mitochondria are critical for the

execution of cell death, and so this pathway has been referred to as the mitochondrial cell

death pathway.


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3.1.1. Mitocondrial Membrane Polarization (∆ψ)

Mitochondrial membrane permeabilization (MMP) is frequently the decisive event that

delimits the frontier between survival and death. Local players that determine the propensity to

MMP include the pro- and antiapoptotic members of the B-cell lymphoma (Bcl-2) family,

proteins from the mitochondrial permeability transition pore complex, as well as a plethora of

interacting partners including mitochondrial lipids. The inhibition of MMP constitutes an

important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely,

induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy [3].

MMP is characterized by several hallmarks that include:

1) the release of cytochrome c (cyt-c), second mitochondria-derived activator of caspase/direct

inhibitor of apoptosis binding protein (IAP) with a low isoelectric point (pI) (Smac/DIABLO),

through the mitochondrial outer membrane and the subsequent activation of effector caspases;

2) the release of caspase-independent apoptogenic death effectors, such as apoptosis inducing

factor (AIF) and endonuclease G (EndoG);

3) a bioenergetic catastrophe including the arrest of oxidative phosphorylation, as well as the

accumulation of reactive oxygen species (ROS),

4) an alteration of the ∆ψ of the mitochondrial inner membrane.

According to current knowledge, the principal mechanism leading to inner membrane

permeabilization is the so-called “permeability transition.”

3.1.2. The mitochondrial permeability transition (mPT)

The mitochondrial permeability transition (mPT) refers to a transition in the permeability

of the mitochondrial inner membrane that occurs when mitochondria in vitro are treated with

calcium [Ca2+] or reagents that increase oxidative stress [4-10].


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Fig.1. PTPC architecture [11]. Although the exact molecular composition of the permeability transition pore complex (PTPC) has not been clearly established yet, a consensus has started to emerge about the proteins involved in its scaffold structure, which builds up at the contact sites between the mitochondrial outer and inner membranes (OM and IM). In addition to the VDAC and the adenine nucleotide translocase (ANT), which represent the main PTPC components, these include hexokinase (HK, interacting with VDAC from the cytosol), creatine kinase (CK, interacting with PTPC from the intermembrane space, IMS), peripheral-type benzodiazepine receptor (PBR, interacting with PTPC from OM), and cyclophilin D (CypD, interacting with ANT from the mitochondrial matrix). HK and CK seemingly associate with the PTPC scaffold in a mutually exclusive fashion. Both anti- and proapoptotic members of the Bcl-2 family modulate the activity of PTPC, through direct interactions with ANT or VDAC.

The exact molecular nature of the permeability transition pore, the PTP is still a matter of

debate, although an emerging consensus considers a multicomponent protein complex, the

PTPC, and not a single protein, as being responsible for the opening of PTP. PTPC is

assembled at the contact sites of the mitochondrial membranes and that its scaffold structure is

based on the dynamic interaction between VDAC, ANT, and CypD [1, 10, 12]. The voltage-

dependent anionic channel, VDAC protein is a pore in the mitochondrial outer membrane that

allows low molecular weight solutes to pass and gain access to inner membrane transport

systems. The adenine nucleotide translocator, ANT is a structural component of the MPT, and

matrix cyclophilin D (CyP-D) bound to complexes of VDAC and the ANT to form the MPT

[7].


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3.1.3. BH3 domain proteins, the Bcl-2 family

BH3 domain-only proteins play a significant role in the processes of cell death and

survival [13-16]. Mammalian cells express a group of evolutionarily conserved proteins

known as the Bcl-2 family. The effect of Bcl-2 proteins on the apoptotic process is due to the

presence of one or more conserved regions of amino acid sequences, known as Bcl-2

homology (BH) domains. Based on their function, Bcl-2 proteins can have either proapoptotic

(Bax, Bad, Bak, Bim, and Bid) or antiapoptotic (Bcl-2 and Bcl-xL) properties.

Fig.2. The mammalian Bcl-2 family members [17].

All antiapoptotic Bcl-2- related proteins described to date contain BH1, BH2, and

sometimes BH4 domains in addition to the BH3 domain [18-20]. Proapoptotic family

members either share the multidomain structure (e.g. Bax and Bak) or contain only the BH3

domain (e.g. Bim and Bid). Bcl-2 proteins containing only the BH3 domain have been

suggested to play an important role in initiating mitochondrial-mediated apoptosis [21-23].


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Proapoptotic BH3 domain-containing proteins are generally regarded as latent death factors

that are normally held in check and must be activated to exhibit their death-inducing functions.

Several mechanisms appear to contribute to the activation of the prodeath function of Bcl-2

family proteins. Bax undergoes conformational changes, relocates to mitochondria, may

oligomerize with other Bax molecules in the mitochondrial membrane, and can be cleaved by

calpain to enhance its proapoptotic effects [24-28]. Bid and Bim share a common mode of

action via BH3-domain-mediated binding to Bax-type proteins at the outer mitochondrial

membrane [26]. This physical interaction is believed to trigger a conformational change of the

multidomain proapoptotic members, resulting in their intramembranous oligomerization and

permeabilization of the outer mitochondrial membrane [27]. In addition, interactions between

proapoptotic Bcl-2 family members and lipid bilayers have an important contribution to this

process. Specific lipids can promote the membrane association of activated forms of Bax and

Bid and can induce mitochondrial cyt-c release [29]. Specifically, cardiolipin increases binding

of both cBid and tBid to pure lipid vesicles as well as to the outer mitochondrial membranes

[30], and myristoylation of tBid further enhances its membrane avidity [31]. Furthermore,

other possible mechanisms of action have been proposed for proapoptotic BH3 domain-only

proteins, including (i) binding to and neutralization or reversal of prosurvival Bcl-2-type

family member functions [13] and (ii) modulation of resident mitochondrial channels, such as

VDAC and ANT [32]. It was demonstrated that VDACs are not an absolutely necessary

component of the mPT complex [33], but when VDAC is present, it can play a role in the

regulation of mPT [34-36]. There are data indicating that proapoptotic Bcl-2 homologues can

activate mPT in oxidative stress showing that an oxidant-damaged mitochondrial membrane

system can react differently to BH3 domain proteins [36-39]. In addition, the oligomer Bax

alone can induce mitochondrial permeability transition and complete cyt-c release without

oxidative stress, indicating that under certain conditions, BH3 domain proteins can contribute

to mitochondrial inner membrane permeabilization, mPT, and necrotic death [35-40].


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Fig.3. The molecular basis of apoptosis [41].

It was also demonstrated that antiapoptotic Bcl-xL can bind to the VDAC-1 barrel

laterally at strands 17 and 18 and can influence mitochondrial permeabilization [42-44].

Therefore, antiapoptotic Bcl-2 protein can also influence (protect) the inner mitochondrial

system, probably via interaction with VDAC [42-43, 45-46].

3.2. The TIP47 protein

Tail-interacting protein (TIP47) is a member of the perilipin/ adipophilin/TIP47 (PAT)

family, which also includes perilipin, adipose differentiation-related protein (ADRP), S3-12

and OXPAT proteins [47-53]. PAT family surface proteins are components of the plasma

membrane and they influence cellular lipid metabolism and also insulin signaling, by means of

controlling access of lipases to stored lipid esters [52]. PAT proteins are characterized by

primary sequence homology, which is evolutionary conserved across all species [54]. The

distribution of PAT proteins varies among different tissues [48]: while the expression of


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perilipin and S3-12 proteins is restricted to adipose and steroidogenic tissues, ADRP and

TIP47 are found ubiquitously [47, 55-57]. The critical role of PAT proteins in the packaging

and storage of neutral lipids and as components of lipid droplets has been established by a

number of studies [49, 52, 57-62].

TIP47 was primarily identified as a possible cargo protein involved in the delivery of

mannose 6-phosphate receptors (M6PR) from endosomes to the trans-Golgi network [63-64].

It was shown to bind to the cytoplasmic domains of cation-independent and cation-dependent

M6PR [65-66] and to be transported to late endosomes by binding to Rab9 GTPase [67-68].

The recognition of a sequence homology between ADRP and TIP47 [57, 69] led to the

discovery that TIP47, like other PAT family members, associates with lipid droplets [57].

Although this finding was initially questioned [58], the role of TIP47 in lipid metabolism was

subsequently confirmed by various sources [49, 54, 61]. Besides its apparent function in lipid

metabolism, TIP47, along with other PAT proteins, influences insulin signaling [52]. The

combined knockout of ADRP and TIP47 was shown to lead to decreased insulin sensitivity

[70-71]. A recent study reported the possible role of TIP47 in Human Immunodeficiency Virus

(HIV-1) infection [72]. TIP47, as a cellular cofactor, was found to play a part in Env

incorporation, allowing the encounter and physical association between HIV-1 Gag and Env

proteins during the viral assembly process [72].

Parallel to studies conducted by other laboratories, our work-group carried out

experiments to elucidate the role of TIP47, which shed new light on the function of this

controversial protein. TIP47 was found to be identical to a soluble, pregnancy-related placental

tissue protein 17b (PP17b) [73]. Of particular significance was the discovery, that, compared

to normal cervical tissue, TIP47 is overexpressed in cervix carcinoma, as is its messenger

RNA in the HeLa cancer cell line [73-75]. Moreover, a correlation could be detected between

the level of TIP47 expression and the different stages of cervical intraepithelial neoplasia

(CIN) [76]. Moderate immunohistochemical staining of the dysplastic cells was apparent in

CIN I-II, whereas strong staining was observed in CIN III and in situ carcinoma [76].

Recently, the role of TIP47 as a potential tumor marker of cervical carcinoma was suggested

[77]. Since TIP47 is secreted into the serum, and its level is elevated in the progression of the

tumor to invasive or metastatic carcinoma and mitigated after treatment, the monitoring of

TIP47 by immunological methods may be beneficial for cervical cancer patients [77].

However, to this date, there is no data available for its possible role in tumor

development, or in regulation of cell death.


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3.3. SOUL/HBP2, a novel BH3 domain protein

Role of heme-containing proteins in the regulation of cell death and survival are well

characterized. They can affect formation of reactive oxygen species hence induction of direct

oxidative damages as well as induction of mitochondrial permeability transition (mPT) [78-

79].

Heme-binding protein 1 (HBP22) is an ubiquitously expressed protein having high

affinity for heme and protoporphyrine [79-80]. Human SOUL (SOUL) is a 23 kDa haem-

binding protein that was first identified as the PP23 protein isolated from human full-term

placentas. [79-80]. SOUL is expressed just in some specific tissues, has more than 40%

sequence homology with HBP22, and has much higher binding affinity for porphyrines than

HBP22 [81].

SOUL facilitates mitochondrial permeability transition and cell death without affecting

reactive oxygen production [79]. The data base search indicated that heme-binding protein

2/SOUL has a BH3 domain-like structure. The presence of a BH3 domain in SOUL and the

above data indicate that this protein besides binding heme may have a role in the processes of

cell death and survival. In present we provide evidence for the sensitization effect of SOUL in

hydrogen-peroxide-induced cell death, the facilitation of the release of pro-apoptotic

mitochondrial proteins and the promotion of the collapse of MMP both in living cells and in

vitro in isolated mitochondria. We show that SOUL promote the permeabilization of both

outer and inner mitochondrial membranes in oxidative stress and its effect can be reversed by

deleting the putative BH3 sequence from SOUL as well as by inhibition of mitochondrial

permeability transition either by cypD suppression or overexpression of anti-apoptotic Bcl-2

members.

SOUL can facilitate cell death in Taxol and A23187-treated cells [79]. Taxol is a

mitotic inhibitor used in cancer chemotherapy. Paclitaxel stabilizes microtubules and as a

result, interferes with the normal breakdown of microtubules during cell division [82-88].

A23187 is a mobile ion-carrier that forms stable complexes with divalent cations (ions with a

charge of +2). A23187 is also known as Calcium ionophore. The ionophore is used to increase

intracellular Ca2+ levels in intact cells and to induce apoptosis in some cells [89].

Analyzing the protein sequence data base, we found that SOUL contained a sequence

with a high similarity to BH3 domains, suggesting that this feature may account for its


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observed cell death-facilitating effect. Previously, SOUL was reported to be strongly

expressed in retina, the pineal gland, and liver [90-91]. We found that it was expressed in

various tissues to different extents.


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4. Aims of the study

The aim of this work was to further clarify the function of TIP47 protein. In addition,

we investigated interactions of SOUL, a novel BH3 domain protein between Bcl-2 family

members in oxidative stress.

The present study is aimed at a more in-depth study of these two regulator proteins,

including:

• To describe the effect of TIP47 on cell death by measuring cell viability of TIP47

overexpressing and control cells treated by different types of cell death inducing agents

• To show the effect of TIP47 suppression by siRNA technique on cell death in

overexpressing and control cells using a Hela cell modell

• To analyse in role of TIP47 of both apoptotic and necrotic cell death

• To find intracellular localization of TIP47 in oxidative stress

• To describe the effect of TIP47 on the mitochondrial permeability transition and on the

stability of the Mitochondrial Membrane Potential (∆ψ)

• To elucidate the effect of TIP47 on ROS-production in TIP47 overexpressing and

control cells

• To find the effect of TIP47 on the pro- and anti-apoptotic signaling pathways

• To show the effect of deletion of the putative BH3 domain on the cell death

• To determine of the role of mPT complex in the SOUL-induced sensitization toward

oxidative stress

• To describe the effect of SOUL on cell death is inhibited by the overexpression of anti-

apoptotic proteins

• To describe the effect of SOUL on the hydrogen-peroxide-induced collapse of MMP


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5. Materials and methods

5.1. Chemicals and antibodies

All the chemicals for cell culture, β-D-1-thiogalactopyranoside (IPTG), Glutathione

Sepharose 4B, methylthiazolyldiphenyl-tetrazolium bromide (MTT), propidium iodide (PI)

and rhodamine-1,2,3 (Rh-123), hydrogen-peroxide (H2O2) were purchased from Sigma-

Aldrich Kft (Budapest, Hungary), Taxol (paclitaxel) was obtained from ICN Biomedicals Inc.

(Aurora, OH). Fluorescent dyes 5,5’,6,6’-tetracloro-1,1’,3,3’-tetraethyl-

benzimidazolylcarbocyanine iodide (JC-1) and fluorescein isothiocyanate (FITC)-conjugated

Annexin V were purchased from Molecular probes (Leiden, Netherlands). The antibodies for

Western blots were purchased as follows: anti-Bax, anti-Bcl-2 and anti-Caspase-3 from Cell

Signaling Technology (Beverly, MA); anti rabbit IgG from Sigma-Aldrich Kft (Budapest,

Hungary), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Delta Biolabs

(Gilroy, CA); anti-AIF and anti-endonuclease G polyclonal antibodies from Oncogene (San

Diego, CA). Mammalian vector pcDNA3.1 was obtained from Invitrogen (Carlsbad, CA,

USA), pEGFP-C1 vector was from BD Biosciences (Franklin Lakes, NJ, USA), Bcl-2-

overexpressing clones were ordered from RZPD GmbH (Berlin, Germany) and pEGFP-C1

vector was purchased from BD Biosciences (Franklin Lakes, NJ, USA). ∆BH3-SOUL plasmid

was ordered from MrGene.com (Mr. Gene GmbH, Regensburg, Germany).

5.2. Preparation of polyclonal antibodies

Two rabbits were immunized subcutaneously at multiple sites with 100 pg of

recombinant TIP47/Glutation-S-transferase (GST) or SOUL/GST fusion protein (with GST

located at the N-terminal of the fusion protein) in Freund's complete adjuvant (Sigma). Four

subsequent booster injections at 4-week intervals were given with 50 pg of protein in Freund's

incomplete adjuvant (Sigma). Blood was collected 14 days after boosting, and the antiserums

were stored at −20˚C. The IgG fraction was isolated from the sera by protein G-Sepharose

chromatography and affinity purified using recombinant TIP47 or SOUL protein bound to a

CNBr-activated Sepharose 4B column and affinity chromatography was carried out as

described previously [92].


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5.3. Construction of expression plasmids, expression and purification

The whole open reading frame of TIP47, t-TIP47 and SOUL were cloned into pGEX-

4T-1 expression vector (Pharmacia, Uppsala, Sweden). The TIP47-, t-TIP47-, SOUL-pGEX-

4T-1 expression vectors were first transformed into E. coli DH5α then into E. coli BL21

competent host strain. Bacteria were induced with isopropyl-b-D-thiogalactopyranoside, and

the expressed TIP47-GST, t-TIP47-GST or SOUL-GST fusion proteins were subsequently

purified with Glutathione Sepharose 4B column (Pharmacia) in the presence of glutathione.

∆BH3-SOUL was cloned into pcDNA3.1.

5.4. Stable transfection

Full length TIP47 cDNA, t-TIP47 cDNA, SOUL cDNA and ∆BH3-SOUL cDNA were

PCR amplified and the construct was subcloned into a pcDNA3.1 (Invitrogen) mammalian

vector containing a Geneticin (G-418) resistance gene. The vectors containing TIP47, t-TIP47,

SOUL, ∆BH3-SOUL or the empty pcDNA3.1 or pEGFP-C1 (BD Biosciences) vector was

transfected into NIH3T3 cells with Lipofectamin 2000 according to the manufacturer’s

protocol (Invitrogen). Cells were then grown in selective media (10% FCS-DMEM containing

500µg/ml G-418). Cell clones were subsequently screened by Western blot analysis for an

increase in TIP47 or SOUL protein expression relative to that in the pcDNA-transfected cells.

TIP47-, t-TIP47-, SOUL- or ∆BH3-SOUL overexpressing clones were selected for further

experiments.

5.5. Cell culture and sample preparation

NIH3T3 and HeLa cells were purchased from the American Type Culture Collection

(Manassas, VA). All cell lines were maintained in Dulbecco's Modified Eagle Medium

(DMEM) supplemented with 10% FCS, 2 units/ml penicillin-streptomycin mixture (Flow

Laboratories, Rockville, MD) and incubated in 5% CO2 - 95% air at 37°C. Cells were

harvested and low-speed centrifuged, then the pellet was dispersed by vortexing in lysis buffer


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(50 mM Tris pH 7.4, 1 mM PMSF) for 10 min at 4ºC. After further cell disruption in a

Teflon/glass homogenizer, the homogenate was pelleted, and the supernatant was measured by

BCA reagent and equalized for 1 mg/ml protein content in Laemmli solution for Western

blotting.

Isolation of subcellular fractions (cytosol, nuclear and mitochondrial fractions) was

carried out by standard lab protocols as described previously [93-94].

5.6. Western blot analysis

100 ng/lane protein of cell culture or mitochondrion samples were subjected to 12 %

(w/v) SDS/PAGE (BioRad, Hercules, Ca, USA). Immunoblots were blocked, and developed

with polyclonal anti-TIP47 or anti-SOUL antibody [77] or polyclonal antibodies against the

cytosolic marker glycerinaldehyde-3-phosphate dehydrogenase (GA3PD), the mitochondrial

marker pyruvate decarboxylase 1α (PDC-1α) and the nuclear marker histon H1 (Histon H1), or

polyclonal antibodies against the signal transduction pathway proteins and horseradish-

peroxidase-labeled secondary IgG as described in [94]. Protein bands were revealed by ECL

chemiluminescence system. All experiments were repeated four times.

5.7. Suppression of TIP47 expression by diced siRNA technique

The whole coding sequence of TIP47 was Polymerase Chain Reaction (PCR)

amplified, double-stranded RNA (dsRNA) was generated by the BLOCK-IT RNAi TOPO

Transcription KIT, the dsRNA was diced by BLOCK-IT Dicer RNAi Transfection KIT and

the result was dsiRNA. The whole procedure was done according to the manufacturer’s

manual (Invitrogen). HeLa cells were transiently transfected with the dsiRNA in OPTI-MEM I

Reduced Serum Medium (Invitrogen) using Lipofectamine 2000 (Invitrogen). The effect of

suppression was tested by Western blot using polyclonal anti-TIP47 primary antibody.


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5.8. Cell viability assay

Cells were seeded into 96-well plates at a starting density of 104cells/well and cultured

overnight before H2O2 or Taxol was added to the medium at a concentration and composition

indicated in the figure legends. After the incubation period, the media were removed and

replaced with DMEM containing an appropriate amount of the MTT solution (Chemicon Inc.,

El Segundo, CA) to each well for 4h. The MTT reaction was terminated by adding HCl to the

medium at a final concentration of 10 mM. The amount of water-insoluble blue formasan dye

formed from MTT was proportional to the number of live cells and was determined with an

Anthos Labtech 200 enzyme-linked immunosorbent assay reader at 550 nm wavelength after

dissolving the blue formasan precipitate in 10% SDS. All experiments were run in at least four

parallels and repeated three times.

5.9. Determination of intracellular reactive oxygen species (ROS)

Intracellular ROS were determined using the oxidation-sensitive C-400 fluorescent

dye. Cells were seeded into 96-well plates at a starting density of 104 cell/well and cultured

overnight, and then were subjected to 0, 0.5, 0.8, 1.0 or 1.5 mM H2O2 for 3 h. Cells were

rinsed, labelled with the fluorescent dye for 15 min at 37 ºC, and analyzed using Anthos

Labtech 200 enzyme-linked immunosorbent assay reader at 485 or 578 nm excitation and 555

or 597 nm emission wavelengths for C-400.

5.10. Detecting mitochondrial membrane potential (∆ψ)

The changes in ∆ψ were assayed using JC-1 dye, which is taken up by the

mitochondria. Empty pcDNA-, t-TIP47-, TIP47-, Bcl-2- SOUL- or ∆BH3-SOUL- transfected

NIH3T3 cells were seeded at 1 x 106 cells/well in a 6-well plate containing coverslips and

cultured at least overnight before the experiment. After subjecting the cells to the appropriate

treatment (indicated in the figure legends), the coverslips were rinsed twice in PBS then placed

upside down on the top of a small chamber formed by a microscope slide, filled with PBS

supplemented with 0.5% FCS and containing 5 µg/ml JC-1-dye (Molecular probes). Cells


20

were imaged with a Zeiss Axiovert 25 fluorescent microscope equipped with a ProgRes C12

Plus CCD camera using a 63 x objective and epifluorescent illumination. For JC-1

fluorescence, the cells were loaded with the dye for 15 min, then the same microscopic field

was imaged first with 546 nm bandpass excitation and 590 nm emission (red), then with green

filters. Under these conditions we did not observe considerable bleed-through between the red

and green images.

5.11. Isolation of mitochondria for the in vitro study

Rats were sacrificed and the mitochondria were isolated from the liver by differential

centrifugation as described by a standard protocol [95]. All isolated mitochondria were

purified by Percoll gradient centrifuging [96], and the mitochondrial protein concentrations

were determined by Biuret method with bovine serum albumin as standard. Mitochondrial

membrane potential was monitored by fluorescence of Rh-123, released from the mitochondria

following the induction of mitochondrial permeability transition (mPT) at room temperature

using a Perkin-Elmer fluorimeter (London, UK) at an excitation wavelength of 495 and an

emission wavelength of 535 nm. Briefly, mitochondria at the concentration of 1 mg protein/ml

were preincubated in the assay buffer (70 mM sucrose, 214 mM mannitol, 20 mM N-2-

hydroxyethyl piperasine-N'-2-ethanesulfonic acid, 5 mM glutamate, 0.5 mM malate, 0.5 mM

phosphate) containing 1 µM Rh-123 and recombinant TIP47 for 60 seconds. Alteration of the

∆ψ was induced by the addition of calcium at the indicated concentration. Changes of

fluorescence intensity were detected for 4 min. The results are demonstrated by representative

original registration curves from five independent experiments, each repeated three times.

5.12. Annexin V-FITC assay

Fluorescent microscopy was performed to visualize the early and late apoptotic cells.

H2O2 treated cells were resuspended in binding buffer and stained with annexin V-FITC/PI for

10-10 min, the cells were then mounted over glass slide and observed under fluorescent

microscope. The same microscopic field was imaged first with 546 nm bandpass excitation

and 590 nm emission (red), then with green filters.


21

5.13. Flow cytometry analysis

Cell death was induced with 500 µM hydrogen peroxide (Sigma) for 3 hours in sham-

transfected, t-TIP47-, TIP47- or SOUL-overexpressing NIH3T3 cells. SOUL-overexpressing

NIH3T3 cells were either co-overexpressing or not Bcl-2 or Bcl-xL proteins. After treatment,

cells were rinsed and harvested. The annexin V FLUOS staining kit (Roche Applied Science)

was used to stain cells with fluorescent isothiocyanate-conjugated annexin V and PI according

to the manufacturer’s protocol. The cells were analyzed by flow cytometry on a BD

FacsCalibur flow cytometer (BD Biosciences). Quadrant dot plots were created by Cellquest

software (BD Biosciences) to identify living and necrotic cells and cells in the early or late

phase of apoptosis [97]. Cells in each category are expressed as a percentage of the total

number of stained cells counted and are presented as pie charts.

5.14. Statistical analysis

Each experiment was repeated at least three times. Values in the figures, tables and text

are expressed as mean ± SEM of n observations. Statistical analysis was performed by analysis

of variance followed by Student’s t-test. Statistical significance was set at p<0,05.


22

6. Results

6.1. TIP47 protects mitochondrial membrane integrity and inhibits cell

death

6.1.1. Effect of expression of TIP47 on H2O2- or Taxol-induced cell death

To evaluate possible physiological functions of TIP47, we transfected NIH3T3 cells

that endogenously express TIP47 at low extent with empty pcDNA3.1 vector, pEGFP-C1 and

a construct with pcDNA3.1 containing the full-length TIP47 or t-TIP47 cDNA. Effectiveness

of the transfection was assessed by immunoblotting utilizing a custom- made anti-TIP47

primary antibody [77].

Transfected cells were treated with 0-500 µM H2O2 (Fig.5A) or 0-1000 nM Taxol

(Fig.5B) for 24hours. Cell viabilities were measured by MTT assay and values were presented

in a bar diagram.

TIP47 overexpression significantly increased TIP47 levels (Fig.4) and the resistance of

NIH3T3 cells against hydrogen peroxide induced cell death (Fig.5A) or Taxol induced cell

death (Fig.5B) compared to the mock-, or t-TIP47 - transfected cells and even GFP-

overexpressing cells, which were also used as controls.

- β-actin

- t-TIP47

- TIP47

1: pcDNA2: GFP3: t-TIP474: TIP47

- β-actin

- t-TIP47

- TIP47

1: pcDNA2: GFP3: t-TIP474: TIP47

Fig.4. Level of TIP47 in NIH3T3 cells. Level of TIP47 expression was assessed by immunoblot analysis (A) utilizing custom-made anti-TIP47 primary antibody in mock-transfected (1), GFP-expressing (2), t-TIP47-overexpressing (3) or TIP47-overexpressing (4) NIH3T3 cells.


23

A B

0

20

40

60

80

100

0 10 100 300 500 µM H2O2

Via

bilit

y (%

)

pcDNA

TIP47

t-TIP47

GFP

****

***

0

20

40

60

80

100

0 10 100 300 500 µM H2O2

Via

bilit

y (%

)

pcDNA

TIP47

t-TIP47

GFP

****

***

0

20

40

60

80

100

0 10 100 1000 nM Taxol

Via

bili

ty (

%)

pcDNA

TIP47

t-TIP47

GFP

Fig.5. Effect of TIP47 overexpression on the viability in oxidative stress or Taxol- induced cell death. Mock- (empty bars), TIP47- (light grey bars), t-TIP47- (full bars) and GFP- (dark grey bars) transfected cells were treated with 0-500µM H2O2 (A) or 0-1000 nM Taxol (B) for 24hours. Cell viabilities were measured by MTT assay and values were presented in a bar diagram. Values are expressed as % of the untreated mock-transfected cells, means ± S.E.M. of four independent experiments. Data are expressed as mean ± S.E.M. *P<0.05; ** P<0.01, *** P<0.001 compared to control values unless otherwise indicated.

6.1.2. The effect of H2O2 or Taxol treatment on the cell viability of HeLa cells transfected

with TIP47 siRNA

As an opposite approach, we suppressed TIP47 expression by dsiRNA technique in

HeLa (human cervical carcinoma) cells (Fig.6A) that express TIP47 at high extent

endogenously. Cells were monitored for 3 days. TIP47 expression was detected by

immunoblot analysis using the polyclonal anti-TIP47 antibody. The highest level of TIP47

silencing occurred on the 2nd day after transfection. Control cells were transfected by anti-

LacZ dsiRNA. Untransfected cells, control cells and anti-TIP47 dsiRNA transfected cells were

incubated for two days, then the they were treated with 0-300µM H2O2 (Fig.6B) or 0-1000

nM Taxol (Fig.6C) for 24 hours before determining cell viabilities by MTT assay. As

compared to the mock- transfected wild type cells or the LacZ siRNA control cells TIP47

suppression sensitized the cells to hydrogen peroxide- and Taxol-induced cell death (Fig.6B,

C) supporting the idea that TIP47 might have a cytoprotective role.


24

A

C 1. 2. 3.

- TIP47

- β-actin

C 1. 2. 3.

- TIP47

- β-actin

B C

0

20

40

60

80

100

0 50 100 300 µM H2O2

Via

bilit

y (%

)

Untransfected

TIP47 siRNA

LacZ siRNA

***

**

0

20

40

60

80

100

0 50 100 300 µM H2O2

Via

bilit

y (%

)

Untransfected

TIP47 siRNA

LacZ siRNA

***

**

0

20

40

60

80

100

0 10 100 1000 nM Taxol

Via

bilit

y (%

)

Untransfected

TIP47 siRNA

LacZ siRNA

Fig.6. Effect of TIP47 suppression on the viability in oxidative stress or taxol- induced cell death. TIP47 was silenced by dsiRNA in Hela cells according to the manufacturer’s protocol. Cells were monitored for 3 days, then TIP47 expression was detected by immunoblot analysis using the polyclonal anti-TIP47 antibody (A). Control cells were transfected by anti-LacZ dsiRNA. Untransfected cells (empty bars), anti-TIP47 dsiRNA transfected cells (grey bars) and control cells (dark bars) were incubated for two days, then the they were treated with 0-300µM H2O2 (B) or 0-1000 nM Taxol (C) for 24 hours before determining cell viabilities by MTT assay. Values are expressed as % of the untreated mock-transfected cells, means ± S.E.M. of four independent experiments. Data are expressed as mean ± S.E.M. *P<0.05; ** P<0.01, *** P<0.001 compared to control values unless otherwise indicated.

6.1.3. Induction of apoptosis by H2O2, Taxol and Etoposide in NIH3T3 cells

Using the differential fluorescence between annexin V-5-fluorescein isothiocyanate

(FITC) and propidium iodide, apoptosis and/or necrosis was visualized in transfected cells.

Annexin-V binds to phosphatidylserine residues that appear at the outer surface of the cell

membrane as an early indicator of apoptosis, while the entry of propidium iodide into the cell

indicates loss of membrane integrity and so necrosis. To easily detect the changes present in

apoptosis we treated cells with Etoposide [98]. Etoposide is a cancer drug. It inhibits the

enzyme topoisomerase II, which unwinds DNA, and by doing so causes DNA strands to break.

The untreated cells retained their morphology throughout the procedure (Fig.7A. 1, 2,

3, 4 and Fig.7B. 1, 2, 3) and treated with 500 µM hydrogen-peroxide (Fig.7A. 5, 6, 7), 100


25

nM Taxol (Fig.7B. 4, 5, 6) or 50µM etoposide (Fig.7A. 8) for 3hours. The results show that

after treatment with H2O2 or Taxol necrosis and late apoptosis were found to be induced in the

mock- and t-TIP47 transfected cells (Fig.7A. 5, 7 and Fig.7B. 4, 6). Mock transfected cells

after etoposide treatment or TIP47 overexpressing cells after H2O2 or Taxol treatment showed

only green fluorescence, hence early apoptosis.

A

H2O2

1 2

7

3 4

85 6

etoposide

pcDNA t-TIP47TIP47 pcDNA

H2O2 H2O2

Untreated

Treated

H2O2

1 2

7

3 4

85 6

etoposide


H2O2 H2O2H2O2

1 2

7

3 4

85 6

etoposide


H2O2 H2O2

Untreated

Treated

B

Taxol

pcDNA TIP47 t-TIP47

1 2 3

4 5 6

Untreated

Taxol

pcDNA TIP47 t-TIP47

1 2 3

4 5 6

Untreated

Fig.7. TIP47 overexpression inhibits H2O2- (A) and Taxol-induced (B) late-apoptotic and necrotic cell death and increases cell survival in NIH3T3 cells. Detection of necrosis and apoptosis by fluorescent microscopy following fluorescein-conjugated Annexin V (green) and PI (red) double staining was performed at the end of 3 hours incubation in the presence or absence of 500 µM H2O2 or 100 nM Taxol. Treated cells show early apoptosis (only green fluorescence) or late apoptosis and necrosis (both green and red fluorescence). Representative merged images of three independent experiments are presented.


26

6.1.4. Detection of H2O2 induced apoptosis by flow cytometry

To determine the type of cell death, the cells were monitored (Fig.8A, B) with flow

cytometry. Mock transfected (pcDNA), TIP47- or t-TIP47- transfected NIH3T3 cells were

treated with either 500 µM hydrogen-peroxide or 50µM etoposide for 3 hours and analyzed for

annexin V-FITC/PI binding. Fig.8A and Fig.8B show that 92,7 ± 1,75% of the mock

transfected (pcDNA) untreated cells were intact (double-negative) while 4,5 ± 1,8% of them

were necrotic (PI positive), and 0,77 ± 0,31% of them were in early- (annexin V positive), and

2 ± 0,5% were in late (double-positive) apoptosis. TIP47 overexpression by itself did not

affect cell death. Treatment with etoposide as a positive control of apoptosis [99-100] was

found to induce early apoptosis in 26,625 ± 6,2% of the cells, affinity towards annexin V-

FITC and devoid of PI staining. Treatment with hydrogen-peroxide increased the number of

late apoptotic and necrotic cells among the mock-transfected and t-TIP47 overexpressing cells.

When the TIP47 transfected cells were treated with H2O2, the cells died mainly by early

apoptosis 13,57 ± 1,5%, and the number of late apoptotic and necrotic cells decreased

drastically (10,03 ± 1,8% and 8 ± 2%). These results showed that overexpressed TIP47

reduced cell death in NIH3T3 cells.

B pcDNA TIP47 t-TIP47 pcDNA

H2O2 H2O2 H2O2 etoposide

pcDNA TIP47 t-TIP47 pcDNAA


living

necrosis

late apoptosis

early apoptosis

Untreated

Treated

Treated

Untreated











living

necrosis

late apoptosis

early apoptosis

Untreated

Treated

Treated

Untreated

Fig.8. Visualization of apoptotic cells by fluorescent microscopy and detection of apoptosis by flow cytometry. Pie charts (A) demonstrate the distribution of living (white), necrotic (dark gray), late apoptotic (black) and early apoptotic (light grey) cells. Dot-plots (B) were created from sham- (pcDNA), TIP47- and t-TIP47-transfected cells treated or not with 500 µM H2O2 or 50 µM etoposide for 3h. Horizontal and vertical axes represent annexin V and PI staining intensities. Lower left


27

quadrant; living cells, lower right quadrant; early apoptotic cells, upper left quadrant; necrotic cells, upper right quadrant; late apoptotic cells. Values are means of 3 independent experiments.

6.1.5. Localization of TIP47 in oxidative stress

To demonstrate the redistribution of TIP47 during the cell death process, we performed

immunoblot experiments on fractionated HeLa cells treated or not with 500 µM H2O2 for 3

hours. Fig.9. shows that in untreated HeLa cells, TIP47 was present in the cytoplasm and not

detected in the mitochondrial or the nuclear fraction. However, upon a short exposure to

oxidative stress, there was a significant increase of TIP47 protein in the mitochondrial

subfraction of the cells, accompanied by a slight decrease in the cytosolic fraction. This data

indicate that in response to oxidative stress, TIP47 can relocate to the mitochondria.

C M N C M N

TIP47

Control H 2O2

47 kDa

GA3PD

PDC1αααα

Histon H1

C M N C M N

TIP47

Control H 2O2

47 kDa

GA3PD

PDC1αααα

Histon H1

Fig.9. Intracellular localization of TIP47. Western blot analysis of cytosolic (C) mitochondrial (M) and nuclear (N) fractions of HeLa cell treated (H2O2) or not (Control) with 500 µM H2O2 for 3 hours. Western blotting was developed as described previously using anti-TIP47 primary antibody, the cytosolic marker glycerinaldehyde-3-phosphate dehydrogenase (GA3PD), the mitochondrial marker pyruvate decarboxylase 1α (PDC-1α) and the nuclear marker histon H1 (Histon H1). Photomicrographs demonstrate representative blots of three independent experiments.

6.1.6. Effect of recombinant TIP47 on the mPT and ∆ψ in Vitro

In order to demonstrate the direct effect of TIP47 on mitochondrial permeability

transition, we monitored ∆ψ and mitochondrial swelling from isolated, Percoll gradient-

purified rat liver mitochondria. Supplemented calcium (150 µM) induced the decrease of the

∆ψ, as detected by the release of the membrane potential-sensitive dye, Rh-123, from isolated


28

liver mitochondria (Fig.10A, line “control” ). Adding TIP47 in the 50–400 nM concentration

range increased the ∆ψ and reduced the level of calcium-induced depolarization (Fig.10A, B,

line “TIP47-GST”). Addition of Taxol to mitochondria induced a concentration dependent

depolarization [101]. Under our experimental conditions TIP47, but not its truncated form,

significantly decreased Taxol-induced depolarization showing that TIP47 can directly protect

the mitochondrial membrane system (Fig.10B, line “TIP47-GST” ). These findings were

consistent with our data obtained under in vivo conditions. Swelling of the mitochondria due to

permeability transition was demonstrated by the decrease of the reflectance of 540 nm light. In

isolated liver mitochondria, the swelling induced by 150 µM Ca2+ (Fig.10C, line “control” )

and it was taken as positive control. To detect how the effect of Ca2+ can be blocked we used

cyclosporin A (CsA), which is a well known specific inhibitor of mPT [102]. The effect of 150

µM Ca2+ was completely inhibited by 2.5 µM CsA (Fig.10C, line “control + CsA”) . TIP47-

GST did not have any significant effect on the mitochondrial swelling in the same

concentration range (Fig.10C, line “TIP47-GST”) .

A

170

180

190

200

210

220

230

240

250

Flu

ores

cenc

e in

tens

ity

control

TIP47-GST

GST

t-TIP47-GST+*

1 min.170

180

190

200

210

220

230

240

250

Flu

ores

cenc

e in

tens

ity

control

TIP47-GST

GST

t-TIP47-GST+*

1 min.


29

B

150

160

170

180

190

200

210

220

230

240

250

Flu

ores

cenc

e in

tens

ity

t-TIP47GSTGSTTIP47 GSTTaxolAddition of

proteins

1 min.

0

10

20

30

40

50

0 50 100 200 400 nM

∆ fl

uore

scen

ce in

tens

ity o

f TIP

47-

GS

T h

yper

pola

rizat

ion

0

5

10

15

20

25

TIP47-GST t-TIP47-GST GST

∆ fl

uore

scen

ce in

tens

ity o

f Tax

ol tr

eatm

ent

150

160

170

180

190

200

210

220

230

240

250

Flu

ores

cenc

e in

tens

ity


proteins

1 min.150

160

170

180

190

200

210

220

230

240

250

Flu

ores

cenc

e in

tens

ity


proteins

1 min.

0

10

20

30

40

50

0 50 100 200 400 nM

∆ fl

uore

scen

ce in

tens

ity o

f TIP

47-

GS

T h

yper

pola

rizat

ion

0

5

10

15

20

25

TIP47-GST t-TIP47-GST GST

∆ fl

uore

scen

ce in

tens

ity o

f Tax

ol tr

eatm

ent

C

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

Flu

ores

cenc

e in

tens

ity

c o n tro lc o n tro l + C s AT IP 4 7 -G S TG S Tt-T IP 4 7 -G S T

2 min.

* +

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

4 5 0

5 0 0

Flu

ores

cenc

e in

tens

ity

c o n tro lc o n tro l + C s AT IP 4 7 -G S TG S Tt-T IP 4 7 -G S T

2 min.

* +

Fig.10. Effect of recombinant TIP47 on permeability transition and mitochondrial membrane potential in isolated mitochondria. (A, B) Membrane potential was monitored by measuring the fluorescent intensities of the cationic fluorescent dye Rh-123. Isolated rat liver mitochondria added at the beginning of the measurement took up the dye in a voltage-dependent manner and quenched its fluorescence. Proteins (GST, t-TIP47-GST, TIP47-GST) were loaded at the first arrow (*) and 150 µM Ca2++ (A) or 250 µM Taxol (B) added at the second arrow (+) facilitated depolarization, resulting in release of the dye and an increase of fluorescence intensity. Representative plots of three independent


30

experiments are presented. (C), permeability transition demonstrated by monitoring E540 in isolated rat liver mitochondria was induced by adding 150 µM Ca2++ at the second arrow (+). Separately, 200 nM GST, t-TIP47-GST, TIP47-GST or 2.5 µM CsA were loaded at the first arrow (*). Representative plots of three independent experiments are presented.

6.1.7. The effect of TIP47 on release of mitochondrial intermembrane space proteins

from living cells

Following apoptotic stimuli and the collapse of the mitochondrial membrane potential,

mitochondrial proteins are released into the cytosol or the nucleus where they play a part in the

process of apoptosis. AIF and EndoG, which induce apoptosis in a caspase-independent

manner, translocate to the nucleus, whereas cyt-c, a member of caspase-dependent apoptosis,

is released into the cytosol. The exposure of sham-transfected cells to Taxol resulted in the

intracellular translocation of the mitochondrial proteins, AIF, EndoG and cyt-c. However,

TIP47 overexpression conferred resistance to taxol-induced cell death by inhibiting the

translocation of AIF and EndoG (Fig.11). Interestingly, though, TIP47 overexpression had no

effect on cyt-c release following taxol treatment (data not shown).

1 2 3 4AIF

EndoG

Cytosol

Histon H1

PDC 1α

GAPDH

1: pcDNA

2: pcDNA - taxol treated

3: TIP47

4: TIP47 - taxol treated

Nucleus

Mitochondrion 1 2 3 4

1 2 3 4

AIF

AIF

EndoG

EndoG

Fig.11. TIP47 overexpression inhibited the Taxol-induced nuclear translocation of EndoG and AIF in NIH3T3 cells. Immuno-blot analysis of the pro-apoptotic mitochondrial proteins EndoG and AIF in TIP47-transfected NIH3T3 cells was carried out following a 6 h treatment with taxol at the concentration indicated. Cells transfected with pcDNA served as controls. Even loading was checked by Histon H1, PDC-1α and GAPDH immunoreactivity in the appropriate subcellular fractions. Representative blots of three independent experiments are presented.


31

6.1.8. Effect of TIP47 on the mitochondrial membrane potential in NIH3T3 cells

Depolarization of the mitochondria can be visualized in vivo by using the membrane

potential sensitive dye, JC-1, and fluorescent microscopy. Accumulation and fluorescence of

the dye depends on the mitochondrial membrane potential showing red (590 nm) or green (530

nm) fluorescence when the mitochondria are intact but loosing the red component of the

fluorescence when the mitochondrial membrane potential is decreased. Bcl-2 is involved in the

regulation of apoptosis with complex interactions among the Bcl-2 family members. They

control the signaling events of apoptosis upstream of mitochondrial outer membrane

permeabilization [103]. The antiapoptotic property of Bcl-2 will make transfected cells more

resistant to oxidative stress, so we used it as positive control for our experiment. Mock-, t-

TIP47-, Bcl-2- and TIP47-transfected NIH3T3 cells were treated for 3 hours with 500 µM

hydrogen peroxide or 100 nM Taxol. In mock transfected and t-TIP47-transfected cells,

hydrogen peroxide or Taxol treatment completely abolished the red fluorescence, and the

mitochondria were visible as intense green spots (Fig.12A. 5, 7 and Fig.12B. 5, 7). Untreated,

Bcl-2- and TIP47-overexpressing NIH3T3 cells showed similar mitochondrial staining pattern

as the sham transfected or t-TIP47 transfected cells (Fig.12A. 1, 2, 3, 4 and Fig.12B. 1, 2, 3,

4), but when treated with hydrogen peroxide or Taxol they showed only a small reduction in

the red fluorescence intensity of the mitochondria (Fig.12A. 6, 8 and Fig.12B. 6, 8) indicating

that TIP47 overexpression indeed prevented the hydrogen-peroxide-and Taxol-induced loss of

mitochondrial membrane potential in significant extent.

A

H2O2

1 2 3

5 6 7

4

8

pcDNA t-TIP47TIP47 Bcl-2

untreated

H2O2

1 2 3

5 6 7

4

8

1 2 3

5 6 7

4

8

pcDNA t-TIP47TIP47 Bcl-2

untreated


32

B

1

8765

432

Untreated

Taxol

pcDNA Bcl-2t-TIP47TIP47

1

8765

432

Untreated

Taxol

pcDNA Bcl-2t-TIP47TIP47

Fig.12. Overexpression of TIP47 protected the stability of the mitochondrial membrane potential in H2O2 or Taxol-treated NIH3T3 cells. NIH3T3 cells were transfected with pcDNA, TIP47, t-TIP47 and Bcl-2. Then cells were treated with 500µM H2O2 (A. 5, 6, 7, 8) or 100 nM Taxol (B. 5, 6, 7, 8) for 3 h to induce mitochondrial membrane damage before washing, loading and imaging. Green and red fluorescence images were merged and used to demonstrate the integrity of the mitochondrial membrane potential in JC-1 loaded untreated NIH3T3 cells. Both H2O2 and taxol treatment of cells transfected with TIP47 and Bcl-2 had no effect on mitochondrial membrane potential as visualized by JC-1. Representative images of three independent experiments are presented.

6.1.9. Effect of TIP47 on ROS-production

We used the previously described cells to test the effect of TIP47 on cellular ROS

formation. We found, in agreement with our previous experience, that endogenous ROS

formation is hardly detectable in the untreated cell lines (Fig.13.), so we induced ROS

production by exposing the cells to different concentrations of H2O2 for 3 hours [79]. After

washing off H2O2, we loaded the cells with the non-fluorescent reduced form of fluorescent

redox dye, C-400. The dye became fluorescent as it was oxidized by the intracellular ROS, so

its fluorescence was proportional to the amount of ROS in the cells. Regardless of the pre-

treatment of the cells, we could not detect any significant difference among mock-, t-TIP47-

and TIP47- transfected cells indicating that TIP47 does not affect cellular pro-oxidant levels

and/or ROS-production.


33

Fig.13. ROS formation in cells over-expressing TIP47 protein. ROS formation was assed by measuring the fluorescent intensities of C-400 oxidized by the ROS formed in mock- (empty bars), TIP47- (grey bars) and t-TIP47- (full bars) transfected NIH3T3 cells exposed to 0-1.5 mM H2O2 for 3hours before washing off the H2O2 and loading the cells with the non-fluorescent reduced form of the dyes. Values are expressed in bar diagram in arbitrary units, means ± S.E.M. of six independent experiments.

6.1.10. The effect of TIP47 on the pro- and anti-apoptotic signaling pathways

Following the 100 nM Taxol-treatment of sham-transfected and TIP47-transfected

NIH3T3 cells, we investigated the expression of Bax, Bcl-2 and cleaved caspase-3 proteins. In

cells overexpressing TIP47, elevated levels of anti-apoptotic Bcl-2 protein and diminished

levels of pro-apoptotic Bax and cleaved caspase-3 were detected, indicating anti-apoptotic

qualities gained by cells through TIP47 overexpression. On the other hand, mock-transfected

cells showed increased Bax and cleaved caspase-3 expression and decreased Bcl-2 expression

following 3 hours of taxol treatment (Fig.14.).

C- 400

0

2000

4000

6000

8000

10000

12000

0 500 800 1000 1500 µM H2O2

Flu

ores

cenc

e in

tens

ity

pcDNA

TIP47

t-TIP47


34

Bax

- + - +

β-actin

Bcl-2

Caspase-3

TIP47pcDNA

100 nM Taxol

Total

Cleaved

VDAC

Bax

- + - +

β-actin

Bcl-2

Caspase-3

TIP47pcDNA TIP47pcDNA

100 nM Taxol

Total

Cleaved

VDAC

Fig.14. TIP47 overexpression facilitates the expression of Bcl-2 and suppresses the expression of Bax and cleaved caspase-3 in NIH 3T3 cells. Western blot showing the effect of 100 nm Taxol-treatment on Bcl-2, Bax and cleaved caspase-3 proteins in sham- and TIP47-transfected cells. Cells transfected with pcDNA served as a control. β-actin immunoreactivity was used to show even loading. Representative blots of three independent experiments are presented.


35

6.2. Cell death in oxidative stress by a novel BH3 domain protein

6.2.1. Characterization of SOUL protein

NCBI Database analysis showed that there is a BH3 domain in SOUL protein, which is

a member of the heme binding protein family. A multiple alignment of the BH3 domains of

Bcl-2 associated proteins show high homology to a sequence found in SOUL (Fig.15).

Fig.15. Comparison of the BH3 domains amongst SOUL and Bcl-2 family members. Identical amino acids are black-shaded, dark-gray-shaded amino acids indicate conserved substitutions and light-gray-shaded amino acids mean semi-conserved substitutions. Sequences of Bcl-xL, Bcl-2, Bcl-W, Bim, Bfk, Bik, Bid, Bax, Bad, Bcl-2-KSHV proteins were accessed from NCBI Protein database.

6.2.2. Determination of the role of mPT complex in the SOUL-induced sensitization

toward oxidative stress

We used siRNA to silence cyclophilin D, a constitutional element of the mitochondrial

transition pore. SOUL-overexpressing and sham-transfected control NIH3T3 cells were

pretreated or not with 30 nM cyclophilin D specific siRNA. Efficacy of the suppression of

cyclophilin D and expression of SOUL was tested by Western blotting (Fig 16A). All cells

were treated with different concentrations of hydrogen peroxide ranging from 0 to 500 µM.

We observed that SOUL-overexpressing cells are sensitized to hydrogen-peroxide while the


36

cyclophilin D suppressed cells had similar sensitivity toward it than the sham-transfected cells

(Fig. 16B). Similar experiments were performed with ∆BH3-SOUL overexpressing cells,

however, we did not find any significant difference between these cells and mock-transfected

cells (data not shown). That is, blocking mPT by suppressing cyclophilin D inhibited

mitochondrial permeability transition and blocked the cell death sensitizing effect of SOUL in

oxidative stress. To confirm our findings, we double-stained the above mentioned cells with

Annexin V conjugated FITC and propidium iodide, and used flow cytometer to detect cell

death. Mock-transfected and SOUL-overexpressing NIH3T3 cells were co-transfected (cycD

siRNA) or not (Control) with 30nM cyclophilin D siRNA, and were treated or not with 300

µM hydrogen peroxide for 24 hours (Fig. 16C). Among control, untreated cells, 94.92±3.9%

were intact (annexin V and PI negative), 4.74±2.3% were necrotic (annexin V negative, PI

positive) and 2.08±1.2% were apoptotic (annexin V positive and PI positive or negative). An

increase in the number of necrotic cells (6.74±2.7%) was observed in the SOUL-

overexpressing untreated cells. Treatment with H2O2 increased the number of necrotic cells in

both, the control and in the SOUL-overexpressing cells (44.88±4.1% and 70.42±4.8%,

respectively). When cyclophilin D was silenced, control cells showed basically no change in

survival (90.73±4.2% living). However, unlike in control cells, SOUL overexpression failed to

increase (p=0.17, n=3) percentage of necrotic cells as compared to cyclophilin-D-silenced

mock-transfected cells (41.47±4.8% vs. 36.37±3.2%, respectively) upon H2O2 treatment.

These results indicate that SOUL protein facilitates necrotic cell death in oxidative stress, and

this effect can be specifically inhibited by suppressing cyclophilin D indicating that SOUL

seems to exert its effect by interacting with the mitochondrial permeability transition pore.

A


37

B C

0

25

50

75

100

0 100 300 500 µM H2O2

%

pcDNASOULpcDNA+CycD siRNASOUL+CycD siRNA

Control

CycDsiRNA

pcDNA H2O2 SOUL + H2O2

SOUL

livingnecrosisapop tosis

Control

CycDsiRNA


SOUL

livingnecrosisapop tosis

FIG .16. Suppression of cyclophilin D abolished the effect of SOUL on H2O2-treated NIH3T3 cells. Suppression of cyclophilin D (CycD) was achieved by co-transfecting or not (Control) sham-transfected (pcDNA) and SOUL-expressing (SOUL) NIH3T3 cells with 30 nM specific siRNA-expressing vector (CycD siRNA) according to the manufacturer’s recommendation. The cells were treated for 24 hours with hydrogen peroxide at concentrations ranging from 0 to 500 µM. (A) Demonstration of expression of SOUL and cyclophilin D in the cells was performed by Western blotting utilizing anti-SOUL and anti-cyclophilin D as well as anti-actin (loading control) primary antibodies. Photomicrographs demonstrate representative blots of three independent experiments. (B) Comparison of the effect of H2O2 on survival of NIH3T3 cells transfected with pcDNA3.1 (black bars), SOUL (light gray bars), pcDNA3.1+CycD siRNA (white bars) or SOUL + CycD siRNA (dark grey bars). Survival was measured by the MTT method and was expressed as % of the survival of untreated double sham-transfected cells. Values are means ± S.E.M. of three independent experiments. Significant differences are indicated above the bar; *P<0.01; **P<0.001. (C) Detection of necrosis and apoptosis was by flow cytometry following Fluorescein-conjugated Annexin V and PI double-staining performed at the end of a 24-hour incubation in the presence or absence of 300 µM H2O2. Pie charts demonstrate the distribution of living (white), necrotic (gray) and apoptotic (black) cells. Values are means of 3 independent experiments.

6.2.3. Effect of deletion of the putative BH3 domain on the cell death facilitating effect of

SOUL

Although not exclusively, BH3 domain is required in most of the cases for the function

of the BH3-only proteins. In order to determine the role of the putative BH3 domain we found

in SOUL by multiple sequence alignment, we deleted 9 amino acids (LREDGKVFD) between

position 162 and 170 as it was described previously for Bak [104]. Mock-transfected, ∆BH3-

SOUL- and SOUL-expressing cells were treated with different concentrations of hydrogen

peroxide ranging from 0 to 500 µM for 24 h, then viability was measured by the MTT method.

We found comparable expression level for ∆BH3-SOUL and SOUL by Western blotting

utilizing anti-SOUL primary polyclonal antibody (Fig 17A). Fig.17B shows that SOUL-

overexpressing cells are sensitized to hydrogen-peroxide while the cells overexpressing

∆BH3-SOUL had similar sensitivity toward it than the sham-transfected cells. That is, when a


38

sequence that was assumed to be functional in its putative BH3 domain was deleted, SOUL

failed to sensitize H2O2-induced cell death.

A

28 kDa –

pcDNA ∆BH3-SOUL SOUL

28 kDa –

pcDNA ∆BH3-SOUL SOUL

B

0

25

50

75

100

0 100 300 500 µM H 2 O 2

%

pcD NAS OUL

..B H3-S OUL**

**

*�

0

25

50

75

100

0 100 300 500 µM H 2 O 2

%

pcD NAS OUL

..B H3-S OUL**

**

*�

Fig.17. Deletion of putative BH3 domain from SOUL in H2O2-induced oxidative stress. Sham-transfected (pcDNA) ∆BH3-SOUL- and SOUL-expressing NIH3T3 cells (A, B) were treated for 24 hours with hydrogen peroxide at concentrations ranging from 0 to 500 µM (B). (A) Demonstration of expression of ∆BH3-SOUL and SOUL in the cells was performed by Western blotting utilizing anti-SOUL and anti-actin (loading control) primary antibodies. Photomicrographs demonstrate representative blots of three independent experiments. (B) Comparison of the effect of H2O2 on survival of NIH3T3 cells transfected with pcDNA3.1 (black bars), SOUL (white bars) or ∆BH3-SOUL (light gray bars). Survival was measured by the MTT method and was expressed as % of the survival of untreated non-transfected cells. Values are means ± S.E.M. of three independent experiments. Significant differences are indicated above the bar; *P<0.01; **P<0.001.

6.2.4. Effect of SOUL on cell death is inhibited by the overexpression of anti-apoptotic

proteins

NIH3T3 cells were co-transfected or not with vectors expressing whole length open

reading frame of Bcl-2 or Bcl-xL and empty pcDNA or full-length SOUL cDNA containing

pcDNA vector. Western blotting was used to detect the efficacy of the transfection (Fig 18A,

19A). To determine the functional consequences of the co-overexpression of Bcl-2 and SOUL

proteins, we monitored survival of the cells when treated with different concentrations of

hydrogen-peroxide. Bcl-2 overexpression alone resulted in a survival benefit at all hydrogen

peroxide concentration we used. SOUL overexpression significantly increased the cell death in


39

the 0 – 500 µM hydrogen peroxide concentration range (p<0.01) demonstrating that SOUL

facilitated oxidative stress induced cell death. But, when cells were co-overexpressing the anti-

apoptotic Bcl-2 protein and SOUL, the effect of SOUL protein was counteracted, and survival

of these cells were similar to mock-transfected cells (Fig 18B). Similar data were observed

when Bcl-xL protein was used instead of Bcl-2 in an identical experimental setup (Fig 19). In

the presence of overexpressed Bcl-xL, SOUL could not decrease the percentage of surviving

cells compared to controls (Fig. 19B). Similar experiments were performed with ∆BH3-SOUL

overexpressing cells, however, we did not find any significant difference between these cells

and mock-transfected cells (data not shown). Data from flow cytometry (Fig. 18C, 19C)

following Annexin V-FITC and PI double staining supported our findings, and showed that the

cell death facilitated by SOUL was mainly necrotic. When cells were co-overexpressing Bcl-2

or Bcl-xL, overexpressed SOUL could not facilitate necrotic cell death induced by H2O2

(70.42±4.8% vs. 28.02±1.8% or 36.69±2.5% respectively) while mock-transfected cells

showed virtually no change in survival (96.11±4.7% and 95.28±3.8% living respectively).

These results indicate that SOUL promoted necrotic cell death induced by oxidative stress, and

this effect was counteracted by the presence of anti-apoptotic Bcl-2 homologues.


40

A

B C

0

25

50

75

100

0 100 300 500 µM H2O2

%

pcDNASOULBcl-2SOUL+Bcl-2

Control

Bcl-2


SOUL

livingnecrosisapoptosis

Control

Bcl-2


SOUL

livingnecrosisapoptosis

Fig.18. Anti-apoptotic BH3 domain protein Bcl-2 abolished the effect of SOUL on H2O2-treated NIH3T3 cells. Bcl-2 was overexpressed by co-transfecting or not (Control) sham-transfected (pcDNA) and SOUL-expressing (SOUL) NIH3T3 cells with Bcl-2-expressing vector (Bcl-2). The cells were treated for 24 hours with hydrogen peroxide at concentrations ranging from 0 to 500 µM. (A) Demonstration of expression of SOUL and Bcl-2 in the cells was performed by Western blotting utilizing anti-SOUL and anti-Bcl-2 as well as anti-actin (loading control) primary antibodies. Photomicrographs demonstrate representative blots of three independent experiments. (B) Comparison of the effect of H2O2 on survival of NIH3T3 cells transfected with pcDNA3.1 (black bars), SOUL (light gray bars), pcDNA3.1 + Bcl-2 (white bars) or SOUL + Bcl-2 (dark grey bars). Survival was measured by the MTT method and was expressed as % of the survival of untreated double sham-transfected cells. Values are means ± S.E.M. of three independent experiments. Significant differences are indicated above the bar; *P<0.01; **P<0.001. (C) Detection of necrosis and apoptosis was by flow cytometry following Fluorescein-conjugated Annexin V and PI double-staining performed at the end of a 24-hour incubation in the presence or absence of 300 µM H2O2. Pie charts demonstrate the distribution of living (white), necrotic (gray) and apoptotic (black) cells. Values are means of 3 independent experiments.


41

A

B C

0

25

50

75

100

0 100 300 500 µM H2O2

%

pcDNASOULBcl-xLSOUL+Bcl-xL

Control

Bcl-xL


SOUL

livingnecros isapoptos is

Control

Bcl-xL


SOUL

livingnecros isapoptos is

Fig.19. Anti-apoptotic BH3 domain protein Bcl-xL abolished the effect of SOUL on H2O2-treated NIH3T3 cells. Bcl-xL was overexpressed by co-transfecting or not (Control) sham-transfected (pcDNA) and SOUL-expressing (SOUL) NIH3T3 cells with Bcl-xL-expressing vector (Bcl-xL). The cells were treated for 24 hours with hydrogen peroxide at concentrations ranging from 0 to 500 µM. (A) Demonstration of expression of SOUL and Bcl-xL in the cells was performed by Western blotting utilizing anti-SOUL and anti-Bcl-xL as well as anti-actin (loading control) primary antibodies. Photomicrographs demonstrate representative blots of three independent experiments. (B) Comparison of the effect of H2O2 on survival of NIH3T3 cells transfected with pcDNA3.1 (black bars), SOUL (light gray bars), pcDNA3.1 + Bcl-xL (white bars) or SOUL + Bcl-xL (dark grey bars). Survival was measured by the MTT method and was expressed as % of the survival of untreated double sham-transfected cells. Values are means ± S.E.M. of three independent experiments. Significant differences are indicated above the bar; *P<0.01; **P<0.001. (C) Detection of necrosis and apoptosis was by flow cytometry following Fluorescein-conjugated Annexin V and PI double-staining performed at the end of a 24-hour incubation in the presence or absence of 300 µM H2O2. Pie charts demonstrate the distribution of living (white), necrotic (gray) and apoptotic (black) cells. Values are means of 3 independent experiments.


42

6.2.5. Effect of SOUL on the hydrogen-peroxide induced collapse of MMP

The specific inhibitory effect of cyclophilin D suppression and overexpressed Bcl-2 or Bcl-

xL proteins on the collapse of mitochondrial membrane potential induced by hydrogen-

peroxide was also determined in SOUL-, ∆BH3-SOUL-overexpressing or mock transfected

NIH3T3 cells using 1 µM JC-1 fluorescent dye [105]. We used hydrogen peroxide

concentration that alone was not able to induce significant decrease in MMP. However, in

SOUL overexpressing cells, loss of red fluorescence of JC-1 indicated a significant decrease in

MMP (Fig. 20). Suppression of cyclophilin D (Fig. 20A) and overexpression of Bcl-2 (Fig.

20B) and Bcl-xL (data not shown) prevented the MMP decreasing effect of SOUL in oxidative

stress. However, JC-1 images of ∆BH3-SOUL overexpressing cells were identical to that of

mock-transfected cells (Fig. 20). These data confirm our previous findings that SOUL

facilitates cell death by interacting with the mPT complex since cyclophilin D is integral

protein of the complex and anti-apoptotic Bcl-2 analogues can inhibit mPT by binding to

VDAC and other components of the complex [36, 45].

A

pcDNA

SOUL

H2O2H2O2 +

CycD siRNA CsAUntreated

∆BH3-SOUL

pcDNA

SOUL

H2O2H2O2 +

CycD siRNA CsAUntreated

∆BH3-SOUL


43

B

H2O2 H2O2 + Bcl-2 Bcl-2

pcDNA

SOUL

Untreated

∆BH3-SOUL

H2O2 H2O2 + Bcl-2 Bcl-2

pcDNA

SOUL

Untreated

∆BH3-SOUL

Fig.20. SOUL depolarizes mitochondrial membrane in vivo in H2O2–treated NIH3T3 cells. Mock-transfected (pcDNA), SOUL- and ∆BH3-SOUL-overexpressing cells were co-transfected or not with vectors expressing cyclophilin D siRNA (A) or Bcl-2 protein (B). All cells were exposed or not to 100 µM hydrogen peroxide for 3 hours, then the medium was replaced to fresh one without any agents and containing 1µM JC-1 membrane-potential sensitive fluorescent dye. After 10 min of loading, green and red fluorescence images of the same field were acquired using a fluorescent microscope equipped with a digital camera. The images were merged to demonstrate depolarization of mitochondrial membrane-potential in vivo indicated by loss of the red component of the merged image. Representative merged images of three independent experiments are presented.


44

7. Discussion

Mitochondria play a key role in the process of both apoptotic and necrotic cell death

[106]. We induced cell death either with etoposide that promotes apoptosis [98], we also used

hydrogen-peroxide or Taxol to induce cell deth with different mechanisms. Taxol, a potent

chemotherapeutic agent and currently applied in the treatment of ovarian, lung and breast

cancer [107], is known to bring on cell death by altering microtubule assembly and arresting

the cell cyle in G2/M phase [88].

Despite the controversy surrounding the main function of TIP47, the

oncodevelopmental significance of this protein has been established TIP47 is strongly

involved in cancer progression [74-77, 108-109]. We hypothesized that its lipid-trafficking-

related function might not play a role in tumor development, so we investigated its possible

effect in the regulation of cell death. We overexpressed TIP47 in NIH3T3 cell line that

produces this protein in a very low extent endogenously and used its spliced variant, the

truncated-TIP47 (t-TIP47 or PP17a) for internal control. t-TIP47 was expressed in placenta

[73] and it lacks 183 amino acids from the N-terminus of TIP47. We found that TIP47 or t-

TIP47 overexpression had no effect on the survival of NIH3T3 cells, but when cells were

exposed to oxidative stress TIP47 overexpression resulted in increased cell viability while t-

TIP47 had no protective function at all (Fig.5A, B). Interestingly, Orlicky et al. reported that

the N-terminal region of TIP47 is responsible for its lipid related function [60].

To prove its specific effect we investigated the endogenous rather than overexpressed

TIP47 on the cell death process in HeLa cell line that expresses TIP47 in a high extent

(Fig6A). Suppression of TIP47 by dsiRNA technique reached it maximum on the second day

after transfection, but we also found, that complete TIP47 depletion could not be reached

[110]. Nevertheless, suppression of TIP47 made cells less viable to oxidative stress (Fig6B, C)

indicating its specific effect on cell death.

To understand the cellular mechanism by which TIP47 protects cells from oxidative

stress we investigated the cell death in detail. We found that in TIP47-overexpressing cells

treated with H2O2 or Taxol less necrotic cell death occur; they rather showed the signs of early

apoptosis similarly to etoposide treated cells while the overexpression of t-TIP47 had no effect

whatsoever (Fig.7A, B, Fig.8).


45

AIF and EndoG can be activated by various stress stimuli, including chemotherapeutic

agents [111-112]. When activated and released from the mitochondria, they are translocated to

the nucleus, where they cause the degradation of DNA [113]. Taxol treatment activated AIF

and Endo G and induced their translocation in mock-transfected fibroblast cells. It is of interest

that, in TIP47-transfected cells treatment with taxol did not alter the intracellular localization

of these proteins (Fig.11).

The type of cell death occurring is related to the strength of the insult. During

mitochondrial permeability transition mitochondrial intermembrane proteins could be released

leading to cytochrome c-triggered apoptosis or in case of the permeability transition pore-

dependent failure of ATP generation to necrosis [106]. Since we found that TIP47 can occur in

the mitochondrial subcellular fraction of H2O2 treated cells (Fig.9) we were concerned about

its effect on mitochondria. In vitro we observed that TIP47 did not induce significant swelling

of mitochondria, but it hyperpolarized the mitochondrial membrane while t-TIP47 evoked no

such effect (Fig.10). By using a mitochondrial membrane potential-sensitive fluorescent dye,

JC-1 in NIH3T3 cells we confirmed that TIP47 could stabilize mitochondrial membrane

potential (Fig.12). Several proteins exist that stabilize the mitochondrial membrane.

Endothelin-1 has been reported to be a potent survival factor against myocardial cell death

[114]. Mpv17l overexpression can also stabilize the mitochondrial membrane, but it also forms

complex with HtrA2 that is capable of sensing and regulating ROS net balance in response to

internal and external stress [115]. Since the increase of ROS generation can result in the

collapse of the inner mitochondrial membrane potential and subsequent membrane

permeabilization with the release of cytochrome c into the cytosol, and so the triggering

cellular apoptosis cascades, we determined the effect of TIP47 on intracellular ROS formation.

Surprisingly, we could not observe any significant difference between controls or t-TIP47

transfected or TIP47-overexpressing cells indicating that TIP47 did not contribute to ROS-

formation (Fig.13.)

According to our result TIP47 can stabilize the mitochondrial membrane and influence

cell death without affecting intracellular ROS-formation. This function of TIP47 might be

related to its N-terminal sequence. Overexpression of TIP47 could be a serious advantage for

tumor cells to grow. As we have earlier shown, there is a correlation between the invasiveness

of a cervical tumor and the amount of TIP47 in the cells and the sera [77].


46

Taxol has been shown to regulate the expression of apoptosis-related proteins,

including Bcl-2, Bax, Bcl-x, p21-waf and tumor necrosis factor alpha (TNF-alpha) [83, 116],

and so, to induce apoptosis [117]. Furthermore, a recent study reported that taxol induces

caspase-independent, AIF-dependent apoptosis in SKOV3 cells [107]. To investigate the effect

of TIP47 on the major apoptotic pathways, TIP47-transfected and mock-transfected NIH3T3

cells were subjected to Taxol treatment, and the levels of Bax, Bcl-2, total caspase-3 and

cleaved caspase-3 were determined by Western blot. The anti-apoptotic effect of Bcl-2,

exerted in part by the inhibition of translocation of Bax to the mitochondria, is well

documented, while Bax and caspase-3 are key participants of the caspase-dependent apoptotic

pathway [118]. Cells transfected with TIP47 acquired an anti-apoptotic quality, since Taxol

exposure in these cells led to increased levels of Bcl-2 and reduced levels of Bax and cleaved

caspase-3, as opposed to the control cells (Fig.14).

In our second study, based on multiple-sequence alignment that indicated that SOUL

has a BH3 domain-like structure, we assumed that the cell deathpromoting effect of SOUL

might be related to this structure (Fig.15). To prove this concept, we disrupted the putative

BH3 domain of SOUL by deleting 9 amino acids from the functional core similarly as it was

done for disrupting BH3 domain function in Bak [104, 119]. We found that the resulting

protein failed to facilitate H2O2-induced cell death. Since cyclophilin D is an essential integral

protein of the PTPC [106, 119], it seems likely that SOUL exerts its effect by interacting with

the complex itself. When cell death was induced under the conditions of cyclophilin D

suppression (Fig.16) or Bcl-2 as well as Bcl-xL overexpression (Figs.18 and 19), BH3-SOUL-

expressing cells did not differ significantly from the sham-transfected ones. Furthermore, it did

not promote H2O2-induced mitochondrial membrane depolarization, as was revealed by JC-1

staining (Fig.20). Because excision of this short sequence resulted in loss of all cell death-

related properties of SOUL, and this sequence showed homology to the BH3 domains of

established Bcl-2 family proteins, SOUL could represent a novel BH3 domain-only protein.

It was reported that BH3 domain proteins, such as the proapoptotic Bcl-2 family

member tBid, by forming a complex with another family member, Bax, can induce cyclophilin

D-independent outer mitochondrial membrane permeabilization and release of proapoptotic

mitochondrial proteins [37]. Oligomer Bax can evoke even mitochondrial permeability

transition and mitochondrial swelling [40, 120]. Despite the accompanying release of


47

proapoptotic mitochondrial proteins, mPT induced by oligomer Bax can lead to cyclophilin D-

sensitive necrotic cell death [40, 120]. Besides tBid and Bax, Bak was reported to bind to

VDAC and facilitate necrotic and apoptotic cell death [119-124]. It is very likely that SOUL as

a BH3 domain-only protein exerts its effect in a similar manner. This is specifically true under

the conditions of oxidative stress where mPT is induced, and both apoptotic and necrotic cell

death pathways can be activated [106].

Antiapoptotic Bcl-2 homologues can bind to VDAC and inhibitmPTand cell death [36,

45, 119, 125]. We determined whether these Bcl-2 homologues can reverse the action of

SOUL. Our data clearly showed that both Bcl-2 and Bcl-xL could prevent SOUL-promoted

cell death (Figs.18 and 19). Therefore, antiapoptotic Bcl-2 proteins counteract the effect of

both SOUL and the proapoptotic Bcl-2 proteins in basically the same manner. Similarly to

oligomer Bax [40], SOUL promoted the permeabilization of not only the outer but also the

inner mitochondrial membrane, and as a consequence, SOUL facilitated predominantly

necrotic cell death.

Previously, it was reported that the recombinant SOUL protein oligomerizes and forms

hexamers in aqueous solution [81]. It is also known that Bax and Bak can oligomerize [126],

and their oligomer forms are very effective in inducing cell death [40, 120]. Therefore, it is

likely that oligomerization plays a role in the effect of SOUL.

The best studied BH3 domain-only proteins, Bim and Bid, permeabilize the outer

mitochondrial membrane and release proapoptotic mitochondrial proteins without affecting the

integrity of the inner membrane; therefore, they induce cyclophilin D-insensitive apoptotic

death [37, 102, 127]. In contrast, SOUL could not induce cell death by itself like Bim and Bid,

but when cell death was induced by various stimuli, such as Taxol, A23187 [79] or H2O2,

SOUL enhanced it and directed it mainly toward necrosis. The effects on the cell death process

of oligomer Bax and SOUL are very similar. Both are capable of permeabilizing the inner

together with the outer mitochondrial membrane, inducing the release of proapoptotic

mitochondrial proteins and promoting cell death. However, Bax can induce both necrotic and

apoptotic cell death by itself [37, 40, 120], whereas SOUL can only facilitate cell death

induced by other stimuli and direct it toward a necrotic mechanism, presumably by amplifying

mitochondrial impairment.


48

All together, we provided evidence that TIP47 and SOUL can affect the cell death

process in a different way.

We proved, that SOUL senzitizes cells to necrotic cell death, not only permeabilized

the outer mitochondrial membrane like proapoptotic Bcl-2 homologues but also facilitated an

inner membrane permeabilization in a cyclophilin D-dependent manner and the collapse of

mitochondrial membrane potential. Most likely, this difference is responsible for the observed

properties of SOUL, namely that it cannot induce cell death alone like other proapoptotic BH3

domain-only proteins but facilitates predominantly necrotic cell death. Thus, SOUL plays a

much more complex role in cell physiology than its mere possible involvement in binding and

transporting heme intermediates.

This study provides convincing evidence of the protective effect of TIP47 which exerts

an anti-apoptotic effect on cell survival by inhibiting the activation of proapoptotic proteins,

by assisting the effect of anti-apoptotic proteins and by the direct stabilization of the

mitochondrial membrane potential. Our findings are of particular importance, since they

propose an explanation for the observed cytostatic resistance against taxol-induced cell death

in cancer cells overexpressing TIP47, and thus, lend further insight into the mechanisms used

by cancer cells to evade chemotherapy.

Using our new results we can better understand how and why would elevated TIP47

levels result in the progression of cervical tumors, but still further studies are required to

exploit this property of the protein in the fight against cancer.


49

8. Conclusions

1. Effect of TIP47 on cell death

We examined effect of TIP47 in different conditions by treating cells with cell death

inducing agents and detecting the living using MTT assay. Hydrogen-peroxide causes

oxidative stress and Taxol affects the microtubule system. In the presence of excess TIP47

cells seemed to have increased resistance against the above mentioned drugs in our

experiment. This effect was statistically significant and was detected with all kinds of drugs

used.

2. Effect of TIP47 suppression by siRNA technique on cell death

Gene silencing is generally used to describe the "switching off" of a gene by a

mechanism other than genetic modification. This process caused by the lack of this specific

protein. When TIP47 was silenced using specific dsiRNAs in cells endogenously expressing it

significant sensitization was observed against the cell death inducing agents as expected.

3. Role of TIP47 in cell death

We used flow cytometry and fluorescent microscopy in this experiment. Cells were

treated with different agent and were stained with propidium iodide and FITC-conjugated

Annexin V to distinguish between the two types of cell death. When we treated the TIP47

transfected cells, the cells died mainly by early apoptosis, and the number of late apoptotic and

necrotic cells decreased drastically. Taxol treatment activated AIF and Endo G and induced

their translocation in mock-transfected fibroblast cells. It is of interest that, in TIP47-

transfected cells treatment with Taxol did not alter the intracellular localization of these

proteins.

4. Intracellular localization of TIP47 in oxidative stress

We detected TIP47’s subcellular localization with Western blot after fractionating cells

endogenously producing high amount of TIP47. According to our results TIP47 was found in


50

the cytoplasm, but after hydrogen peroxide treatment it was also present in the mitochondrial

fraction in a smaller quantity.

5. Effect of TIP47 on the mitochondrial permeability transition and on the

Mitochondrial Membrane Potential (∆ψ)

We provide that TIP47 can bind to mitochondria and protect mitochondrial membrane

integrity, as well as prevent oxidative stress induced cell death. First we tracked the change of

the mitochondrial membrane potential in TIP47-overexpressing NIH3T3 cells using

fluorescent microscopy. In the TIP47 transfected cells the mitochondrial membrane potential

remained stable after treatment. This result reised our interest and we isolated mitochondria

from rat liver to detect direct effect of recombinant TIP47 in vitro.Recombinant TIP47 not

induced mitochondrial permeability transition when we added subthreshold concentrations of

calcium. These data supports our view that TIP47 protects cell by stabilizing mitochondrial

membrane.

6. Effect of TIP47 on ROS-production

We could not observe any significant difference between controls or TIP47-

overexpressing cells indicating that TIP47 did not contribute to ROS-formation.

7. Effect of TIP47 on the pro- and anti-apoptotic signaling pathways

We investigated the effect of TIP47 on cell viability and on the pro- and anti-apoptotic

signaling pathways in cells, following treatment with the chemotherapeutic agent, Taxol. We

found, that TIP47 over-expressed cells increased levels of Bcl-2 and reduced levels of Bax and

cleaved caspase-3, as opposed to the control cells.

8. Role of mPT complex in the SOUL-induced sensitization toward oxidative stress

We observed that SOUL-overexpressing cells are sensitized to hydrogen-peroxide

while the cyclophilin D suppressed cells had similar sensitivity toward it than the sham-

transfected cells. We performed similar experiments with ∆BH3-SOUL overexpressing cells.


51

Blocking mPT by suppressing cyclophilin D inhibited mitochondrial permeability transition

and blocked the cell death sensitizing effect of SOUL in oxidative stress.

9. Effect of deletion of the putative BH3 domain on the cell death

We showed that SOUL promotes the permeabilization of both outer and inner

mitochondrial membranes in oxidative stress and that its effect can be reversed by deleting the

putative BH3 sequence from SOUL. When we deleted a 9 amino acid (LREDGKVFD)

sequence that was assumed to be functional in its putative BH3 domain, SOUL failed to

sensitize H2O2 induced cell death.

10. Effect of SOUL on cell death is inhibited by the overexpression of anti-apoptotic

proteins

When cells were co-overexpressing Bcl-2 or Bcl-xL, overexpressed SOUL could not

facilitate necrotic cell death induced by hydrogen-peroxide. Data from flow cytometry showed

that the cell death facilitated by SOUL was mainly necrotic. SOUL promoted necrotic cell

death induced by oxidative stress, and this effect was counteracted by the presence of anti-

apoptotic Bcl-2 homologues.

11. Effect of SOUL on the H2O2-induced collapse of MMP

SOUL facilitated cell death by the promotion of the collapse of mitochondrial

membrane potential (MMP) in living cells. This protein senzitized the cells to cell death by

interacting with the mPT complex since cyclophilin D is integral protein of the complex and

anti-apoptotic Bcl-2 analogues can inhibit mPT by binding to VDAC and other components of

the complex.


52

9. Acknowledgements

These studies were carried out at the Department of Biochemistry and Medical Chemistry,

Medical School of the University of Pécs between 2006-2010.

I want to express my thanks to my teacher and project leader, Professor Dr. Balázs Sümegi

who managed my studies and gave support and useful advises during my work.

I would like to thank Prof. Dr. Ferenc Gallyas for his tutorial work.

I am grateful to my closest colleagues who assisted me in my experimental studies and

gave a hand with a part of the experiments: Dr. András Szigeti, Dr. Szabolcs Bellyei, Dr.

Boglárka Rácz and Alíz Szabó. I wish to thank all participants of the experiments for their

work, all the doctors, PhD students and assistants of the Institute Biochemistry and Medical

Chemistry participating in the project for their theoretical and technical help.

At last but not least, this work would have never been realized without the support and

help of my family.

This work was supported by AOKKA-34039-1/2009 and 34039-23/2009 as well as

Hungarian Research Grants OTKA 68469, K-73738 and Science, Please! Research Team on

Innovation (SROP-4.2.2/08/1/2008-0011).


53

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mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim. Oncogene, 2002. 21(32): p. 4944-56.

125. Kowaltowski AJ, Vercesi AE, and Fiskum G., Bcl-2 prevents mitochondrial

permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides. Cell Death Differ, 2000. 7(10): p. 903-10.

126. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH., Pro-apoptotic

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63

11. List of Publications

This work is based on the following articles:

1. Eniko Hocsak, Boglarka Racz, Aliz Szabo, Laszlo Mester, Edit Rapolti, Szaniszlo Javor,

Szabolcs Bellyei, Ferenc Gallyas Jr., Balazs Sumegi, Andras Szigeti. TIP47 protects

mitochondrial membrane integrity and inhibits oxidative-stress-induced cell death. FEBS

Lett. 2010 Jul 2;584(13):2953-60.

IF: 3,541 (2009)

Cit.: 0

2. Eniko Hocsak, Boglarka Racz, Aliz Szabo, Eva Pozsgai, Andras Szigeti, Edit Szigeti,

Ferenc Gallyas Jr., Balazs Sumegi, Szaniszlo Javor, Szabolcs Bellyei. TIP47 confers

resistance to taxol- induced cell death by preventing the nuclear translocation of AIF and

Endonuclease G. Eur JCB. in press.

IF: 3,314 (2009)

Cit.: 0

3. Andras Szigeti, Eniko Hocsak, Edit Rapolti, Boglarka Racz, Arpad Boronkai, Eva

Pozsgai, Balazs Debreceni, Zita Bognar, Szabolcs Bellyei, Balazs Sumegi, and Ferenc

Gallyas, Jr. Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and

Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein. J Biol

Chem. 2010 Jan 15;285(3):2140-51.

IF: 5,328 (2009)

Cit.: 0


64

Further publications:

1. Mester L, Szabo A, Atlasz T, Szabadfi K, Reglodi D, Kiss P, Racz B, Tamas A, Gallyas F

Jr, Sumegi B, Hocsak E, Gabriel R, Kovacs K. Protection against chronic hypoperfusion-

induced retinal neurodegeneration by PARP inhibition via activation of PI-3-kinase Akt

pathway and suppression of JNK and p38 MAP kinases. Neurotox Res. 2009 Jul; 16(1):68-

76.

IF: 2,43

Cit.: 1

2. Szigeti A, Minik O, Hocsak E, Pozsgai E, Boronkai A, Farkas R, Balint A, Bodis J,

Sumegi B, Bellyei S.: Preliminary study of TIP47 as a possible new biomarker of cervical

dysplasia and invasive carcinoma. Anticancer Res. 2009; 29: 717-724.

IF: 1,428

Cit.: 1

3. Bellyei S, Szigeti A, Pozsgai E, Boronkai A, Gomori E, Hocsak E, Farkas R, Sumegi B,

Gallyas F Jr.: Preventing apoptotic cell death by a novel small heat shock protein. Eur J

Cell Biol. 2007 Mar; 86(3):161-71.

IF: 3,224

Cit.: 10

4. Bellyei S, Szigeti A, Boronkai A, Pozsgai E, Gomori E, Melegh B, Janaky T, Bognar Z,

Hocsak E, Sumegi B, Gallyas F Jr.: Inhibition of cell death by a novel 16.2 kD heat shock

protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation

pathway. Apoptosis. 2007 Jan; 12(1):97-112.

IF: 3,043

Cit.: 7

5. Szigeti A, Bellyei S, Gasz B, Boronkai A, Hocsak E, Minik O, Bognar Z, Varbiro G,

Sumegi B, Gallyas F Jr.: Induction of necrotic cell death and mitochondrial

permeabilization by heme binding protein 2/SOUL. FEBS Lett. 2006 Nov 27;

580(27):6447-54.

IF: 3,372

Cit.: 3


65

6. Bányai K, Forgách P, Erdélyi K, Martella V, Bogdán A, Hocsák E, Havasi V, Melegh B,

Szucs G.: Identification of the novel lapine rotavirus genotype P[22] from an outbreak of

enteritis in a Hungarian rabbitry. Virus Res. 2005 Nov; 113(2):73-80.

IF: 2,562

Cit.: 7

Total IF: 28,242

Total citations: 29

h-index: 3


66

Abstracts

1. Hocsák Enikő, Rácz Boglárka, Szabó Alíz, Bellyei Szabolcs, Szigeti Edit, Mester László,

ifj. Gallyas Ferenc, Sümegi Balázs, Szigeti András: A TIP47 fehérje a mitokondrium

membrán stabilizálásával részt vesz a mitokondrium mediálta sejthalál szabályozásában,

40. Membrán-Transzport Konferencia, Sümeg, 2010. május 18-21.

2. Hocsák Enikő, Rápolti Edit, Jávor Szaniszló, Rácz Boglárka, Szigeti András, Szabó Alíz,

Sümegi Balázs: A TIP47 fehérje gátolja az oxidatív stressz indukált apoptózist és

nekrózist. 39. Membrán-Transzport Konferencia, Sümeg, 2009. május 19-22.

3. Rápolti Edit, Szigeti András, Boronkai Árpád, Hocsák Enikő, Rácz Boglárka, Szabó Alíz,

Pozsgai Éva, Bognár Zita, Bellyei Szabolcs, Sümegi Balázs, ifj. Gallyas Ferenc: Egy új,

BH3 domén fehérje oxidative stresszben elősegíti a mitokondrium belső és külső

membránjának permeabilizációját. 39. Membrán-Transzport Konferencia, Sümeg, 2009.

május 19-22.

4. Rácz Boglárka, Rápolti Edit, Hocsák Enikő, Gasz Balázs, Gallyas Ferenc Jr., Kiss Péter,

Horváth Gabriella, Tamás Andrea, Lubics Andrea, Tóth Gábor, Sümegi Balázs, Reglődi

Dóra: Effects of pituitary adenylate cyclase activating polypeptide (PACAP) in ischemic

and oxidative stress-induced cardiomyocyte damage. 39. Membrán-Transzport

Konferencia, Sümeg, 2009. május 19-22.

5. Mester László, Szabó Alíz, Atlasz Tamás, Szabadfi Krisztina, Reglődi Dóra, Kiss Péter,

Rácz Boglárka, Tamás Andrea, Ifj. Gallyas Ferenc, Sümegi Balázs, Hocsák Enikő,

Gábriel Róbert, Kovács Krisztina: Protection against chronic hypoperfusion-induced

retinal neurodegeneration by PARP inhibition via activation of PI-3-kinase Akt pathway

and suppression of JNK and p38 MAP kinases. 39. Membrán-Transzport Konferencia,

Sümeg, 2009. május 19-22.

6. Szigeti A., Bellyei Sz., Boronkai Á., Pozsgai É., Rápolti E., Hocsák E., Mester L., Sümegi

B.,:A SOUL-fehérje mitokondriális permeabilitás-tranzíción keresztül nekrotikus sejthalált

indukál. 38. Membrán-Transzport Konferencia, Sümeg, 2008. május 20-23.


67

7. Németh Viktória, Montskó Gegő, Solti Izabella, Vető Sára, Tucsek Zsuzsanna, Hocsák

Enikő, Márk László: Komplex biológiai minták GSH tartalmának meghatározása

ioncsapdás tömegspektrometria segítségével. A Magyar Biokémiai Egyesület 2006. évi

Vándorgyűlése, Pécs, 2006. augusztus 30- szeptember2.

8. Bognár Eszter, Solti Izabella, Németh Viktória, Bartha Éva, Tucsek Zsuzsanna, Vető Sára,

Hocsák Enikő, Nagyné Kiss Gyöngyi, Sümegi Balázs, Berente Zoltán: A glükózfelvétel

és a kapcsolódó intracelluláris jelátviteli utak vizsgálata izolált szívmodellen. A Magyar

Biokémiai Egyesület 2006. évi Vándorgyűlése, Pécs, 2006. augusztus 30- szeptember2.

9. Zsuzsanna Tucsek, Balazs Radnai, Zoltan Szabo, Tamas Dolowschiak, Eniko Hocsak,

Aliz Szabo, Jr. Ferenc Gallyas, Tamas Lorand, and Balazs Sumegi: The effect of the IK11,

an E - 4 - arylidene-3-isochromanone on the Hep G2 human hepatocellular carcinoma cell

line. Semmelweis Symposium – Nitric Oxide and Nitrosative stress in the Cardiovascular

System, Budapest, 2006. okt. 29-31.

10. Balazs Radnai, Zsuzsanna Tucsek, BalazsVeres, Katalin Hanto, Peter Jakus, Balazs

Debreceni, Zita Bognar, Eniko Hocsak, Denes Grasz, Ferenc Gallyas Jr. and Balazs

Sumegi: Effect of a PARP inhibitor, HO-3089 on the gene expression profiles of LPS

stimulated RAW 264,7 murine macrophages. Semmelweis Symposium – Nitric Oxide and

Nitrosative stress in the Cardiovascular System, Budapest, 2006. okt. 29-31.

11. Tucsek Zsuzsanna, Radnai Balázs, Szabó Zoltán, Dolowschiák Tamás, Hocsák Enikő,

Szabó Alíz, Solti Izabella, Bognár Eszter, Vető Sára, ifj.Gallyas Ferenc, Loránd Tamás és

Sümegi Balázs: A PJ34 protektív hatása az IK11 indukálta oxidatív stresszben HepG2

sejtvonalon. 36. Membrán-Transzport Konferencia, Sümeg, 2006. május 23-26.

12. Hocsák Enikő, Bellyei Szabolcs, Szigeti András, Boronkai Árpád, Tucsek Zsuzsanna,

Szabó Alíz, Vető Sára, Berki Tímea, Sümegi Balázs: A PP17B fehérje strukturális és

funkcionális vizsgálatai. 36. Membrán-Transzport Konferencia, Sümeg, 2006. május 23-

26.


68

13. Bellyei Sz., Szigeti A., Boronkai Á., Bognar Z., Hocsák E., Tucsek Zs., Pozsgai É.,

Gallyas F. Jr., Sumegi B.: Egy új 16,2 kDa nagyságú kis molekulasúlyú hő-sokk szerű

fehérje bemutatása. 36. Membrán-Transzport Konferencia, Sümeg, 2006. május 23-26.

14. Boronkai Árpád, Bellyei Szabolcs, Szigeti András, Hocsák Enikő, Tucsek Zsuzsanna,

Sümegi Balázs: A galectin-13 jellemzése, sejthalál indukáló hatása. 36. Membrán-

Transzport Konferencia, Sümeg, 2006. május 23-26.

15. Szigeti András, Bellyei Szabolcs, Boronkai Árpád, Bognár Zita, Gasz Balázs, Szabó

Zoltán, Tucsek Zsuzsanna, Hocsák Enikő, Komlósi Katalin, Várbíró Gábor, Melegh Béla,

Janaky Tamás, Sümegi Balázs, ifj. Gallyas Ferenc: MPTIP1, az első csak BH3 domént

tartalmazó permeability transition-t indukáló fehérje. 36. Membrán-Transzport

Konferencia, Sümeg, 2006. május 23-26.

16. Sara Veto, Izabella Solti, Aliz Szabo, Zsuzsanna Tucsek, Viktoria Nemeth, Eniko Hocsak,

Balazs Veres, Zoltan Berente, Balazs Sumegi: Involvement of Akt/protein kinase B

pathway induction in the protective effect of poly – (ADP-ribose) polymerase 1 inhibition

in endotoxin – induced septic shock. 36. Membrán-Transzport Konferencia, Sümeg, 2006.

május 23-26.

17. Solti Izabella, Bognár Zita, Németh Viktória, Bognár Eszter, Vető Sára, Hocsák Enikő,

Nagyné Kiss Gyöngyi, Szántó Árpád, Várbíró Gábor, Sümegi Balázs: Taxol hatása a

mitokondriumra és a szabadgyök képződésre. 36. Membrán-Transzport Konferencia,

Sümeg, 2006. május 23-26.

18. Aliz Szabo, Zita Bognar, Arpad Szanto, Eniko Hocsak, Katalin Hanto, Edina Pandur,

Judit Nagy, Viktor Poor, Balazs Sumegi: Induction of NFKB dependent COX-2

expresssion in liver cells by amiodarone. 36. Membrán-Transzport Konferencia, Sümeg,

2006. május 23-26.

19. Ferenc Gallyas Jr., Balazs Veres, Balazs Radnai, Eniko Hocsak, Balazs Sumegi:

Significant role of cytoprotective kinase signalling mechanisms in the protective effect of

PARP inhibitors in a murine septic shock model. 30th FEBS Congress - 9th IUBMB

Conference Budapest, Hungary 2nd-7th July, 2005. FEBS J. 2005; 272: 424-425.


69

20. Anita Palfi, Ambrus Toth, Katalin Hanto, Eniko Hocsak, Robert Halmosi, Eszter

Szabados, Kalman Hideg, Balazs Sumegi, Kalman Toth: Contribution of intracellular

signaling cascades to the cardioprotective effect of poly (ADP-ribose) polymerase

inhibitors in myocardial ischemia-reperfusion. 30th FEBS Congress - 9th IUBMB

Conference Budapest, Hungary 2nd-7th July, 2005. FEBS J. 2005; 272: 311-311.

Presentations

1. Hocsák Enikő, Szabó Alíz, Szigeti András, Ifj. Gallyas Ferenc, Sümegi Balázs: A TIP47

fehérje gátolja az oxidatív stressz indukált sejthalált, Biológus Doktoranduszok

Konferenciája, Pécs, 2009. November 12-13.

2. Ifj. Gallyas Ferenc, Hocsák Enikő, Pozsgai Éva, Szigeti András, Bellyei Szabolcs, Sümegi

Balázs: Mitokondriális membrán-permeabilitást szabályozó új mechanizmusok, 39.

Membrán-Transzport Konferencia, Sümeg, 2009. május 19-22.

3. Hocsák Enikő: A SOUL fehérje mitokondriális permeabilitás tranzíción keresztül

nekrotikus sejthalált indukál. PTE Grastyán Endre VII. Országos Interdiszciplináris

Konferencia, Pécs, 2009. március 23 – 25.

4. Hocsák Enikő: A SOUL fehérje mitokondriális permeabilitás tranzíción keresztül

nekrotikus sejthalált indukál. XIV. Korányi Frigyes Tudományos Fórum, Budapest, 2009.

március 26.

5. Szigeti András, Hocsák Enikő, Sümegi Balázs, ifj. Gallyas Ferenc: SOUL: Egy új BH3-

domaint tartalmazó fehérje, amely regulálja a mitokondriális permeabilitás tranzíciós

pórust. A Magyar Biokémiai Egyesület 2008. évi Vándorgyűlése, Szeged, 2008 aug. 31-

szept. 3.


70

6. Hocsák Enikő, Mester László, Szabó Alíz, Krisztina Kovács, Szigeti András, Bellyei

Szabolcs, Sümegi Balázs, Gallyas Ferenc Jr.: Mitokondrium mediálta sejthalál indukciója

egy új BH3 domént tartalmazó fehérjével. Magyar Humángenetikai Társaság VII.

Kongresszusa, Pécs, július 11-13.

7. Mester László, Hocsák Enikő, Szabó Alíz, Krisztina Kovács, Bellyei Szabolcs, Szigeti

András, Boronkai Árpád, Pozsgai Éva, Gallyas Ferenc Jr., Sümegi Balázs: Egy új-16.2 kD-

kis hősokk fehérje azonosítása és a jelátviteli folyamatokra gyakorolt szerepének a

vizsgálata. Magyar Humángenetikai Társaság VII. Kongresszusa, Pécs, július 11-13.

8. Hocsák Enikő, Jávor Szaniszló, Bellyei Szabolcs, Szigeti András, Boronkai Árpád,

Sümegi Balázs: A PP17b/TIP47 fehérje strukturális és funkcionális vizsgálatai. PTE

Grastyán Endre VI. Országos Interdiszciplináris Konferencia, Pécs, 2008. március 26 – 28.

9. Radnai Balázs, Tucsek Zsuzsanna, Hocsák Enikő, Vető Sára, Németh Viktória, Bognár

Eszter, Grász Dénes, Berente Zoltán, ifj.Gallyas Ferenc, Sümegi Balázs: A ferulaldehid

hatása az LPS indukálta endotoxikus sokkra egerekben. A Magyar Biokémiai Egyesület

2006. évi Vándorgyűlése, Pécs, 2006. augusztus 30- szeptember2.

10. Balazs Sumegi, Sara Veto, Zsuzsanna Tucsek, Izabella Solti, Eniko Hocsak, Eszter

Bognar, Aliz Szabo, Ferenc Gallyas Jr.: PARP and inflammatory and kinase pathways:

relevance for endotoxic shock. Semmelweis Symposium – Nitric Oxide and Nitrosative

stress in the Cardiovascular System, Budapest, 2006. okt. 29-31.

11. Sümegi Balázs, Kovács Krisztina, Tapodi Antal, Hantó Katalin, Radnai Balázs, ifj. Gallyas

Ferenc, Hocsák Enikő, Hideg Kálmán: Az ADP-riboziláció szerepe a PI3-kináz, Akt és

MAPk jelátviteli útvonalak szabályozásában. XIII. Sejt és Fejlődésbiológiai Napok, Eger,

2005. ápr. 10-12.

12. Bellyei Sz., Szigeti A., Boronkai Á., Bognár Z., Hocsák E., Tucsek Zs., Pozsgai É.,

Gallyas F. Jr., Sümegi B.:Egy új 16.2 kDa nagyságú kis molekulasúlyú hő-sokk szerű

fehérje bemutatása. 36. Membrán-Transzport Konferencia, Sümeg, 2006. május 23-26.


71

13. Szigeti András, Bellyei Szabolcs, Gasz Balázs, Boronkai Árpád, Hocsák Enikő, Minik

Orsolya, Bognár Zita, Várbíró Gábor, Sümegi Balázs, Ifj. Gallyas Ferenc: Egy, BH3

domén-t tartalmazó, mitokondriális permeability transition-t indukáló fehérje azonosítása.

Magyar Biokémiai Egyesület (MBKE) 2006. évi vándorgyűlése, Pécs, 2006. augusztus 30-

szeptember2

14. Bellyei Sz., Szigeti A., Boronkai Á., Bognár Z., Hocsák E., Pozsgai É., Gallyas F. Jr.,

Sümegi B.:Egy új 16.2 kDa nagyságú kis molekulasúlyú hő-sokk szerű fehérje

funkcionális analízise. Magyar Biokémiai Egyesület (MBKE) 2006. évi vándorgyűlése,

Pécs, 2006. augusztus 30- szeptember2

15. Boronkai Árpád, Bellyei Szabolcs, Szigeti András, Hocsák Enikő, Németh Viktória,

Sümegi Balázs: A galectin-13 indukálta apoptózis molekuláris mechanizmusai. Magyar

Biokémiai Egyesület (MBKE) 2006. évi vándorgyűlése, Pécs, 2006. augusztus 30. -

szeptember 2.

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Identification and characterization of novel regulators in …aok.pte.hu/docs/phd/file/dolgozatok/2010/Hocsak_Eniko_PhD_dolgozat.pdf · Identification and characterization of novel