Ubiquitylation: its role and medical significance

Zoltán LipinszkiInstitute of Biochemistry

October 25, 2017

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

• The proteins of the body are in a dynamic state of synthesis and degradation!– It is thought that we degrade and resynthesize ~3-5% of

our cellular proteins daily.• Paradigm that cellular processes are controlled

mainly by only transcription and translation must be changed.

Cellular homeostasisCellular homeostasis

Why are proteins degraded?

• Quality control– Proteins become denatured/misfolded/damaged

• Elevated temperatures (37°C)– Proteins being synthesized are folded incorrectly

• Regulation of biological pathways– Cell cycle– Receptor mediated endocytosis– Synaptic remodeling

Schoenheimer: proteins are in a dynamic turnover

De Duve: protein degradation in lysosomes

Energy dependence of protein degradation tyrosine aminotransferase

TABLE 1.- Resolution of the ATP-dependent cell-free proteolytic system into complementing activities

Az ATP-függő proteolitikus aktivitás frakcionálása DEAE-cellulóz kromatográfiával

"for the discovery of ubiquitin-mediated protein degradation"

Nobel Prize in Chemistry, 2004

Aaron Ciechanover Avram Hershko Irwin Rose

Discovery timeline of the ubiquitin proteasome system

Cyclin levels fluctuate during cell cycle

Mitotic cyclin destruction box

COOHH2N

Cyclin A

Cyclin B1Cyclin B2

Arg-Thr-Val-Leu-Gly-Val-Ile-Gly-Asp

Arg-Thr-Ala-Leu-Gly-Asp-Ile-Gly-Asn

Arg-Ala-Ala-Leu-Gly-Glu-Ile-Gly-Asn

Ubiquitin

composed of 76 amino acids

found only in eukaryotes

highly conserved

synthesized as a polyprotein

forms a heat-stable compact globular structure

exist either in free form or as attachment to other proteins

serves as a tag that marks proteins for degradation

1-MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF-451-MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIF-451-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-451-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45

46-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-7646-AGKQLEDGRTLADYNIQKESTLHLVLRLRGG-7646-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-7646-AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76

Fission yeast humanGreen pea fruitfly

The structure of ubiquitin is conserved from yeast to human

Ubiquitin is synthesized as a polyprotein

Ubiquitin Ubiquitin Ubiquitin UbiquitinRibosomal protein

Transcription/Translation

Ub C-terminal hydrolase

Ubiquitin is conjugated to proteins

Enzymatic cascade of protein ubiquitylation

Ub: ubiquitin

E1: ubiquitin-activating enzyme

E2: ubiquitin-conjugating enzyme

E3: ubiquitin ligase

UbC OH

O

E3E2 SC S

O

UbC S

O

Ub

E1

ATP AMP

UbC NH

O

UbUb

Ub

Degradation by the 26S proteasome

The ubiquitin system is enormous

The genes of the UPS constitutes ~5% of the genome

• E1’s - 1-2 activating enzymes• E2’s - 10-20 conjugating enzymes• E3’s - 500-800 ubiquitin ligase - drives specificity• DUBs - 100 ubiquitin specific proteases

The hierarchical structure of the ubiquitin system

Subunit S. cerevisiae S. pombe Gerincesek Drosophila APC1 Apc1 Cut4 Apc1/Tsg24 shattered

APC2 Apc2/Rsi1 Apc2 Apc2 DmApc2

APC3 Cdc27 Nuc2 Apc3/Cdc27 mákos

APC4 Apc4 Cut20 Apc4 DmApc4

APC5 Apc5 Apc5 Apc5 ida

APC6 Cdc16 Cut9 Apc6/Cdc16 DmCdc16

APC7 --- --- Apc7 DmApc7

APC8 Cdc23 Cut23 Apc8/Cdc23 DmApc8

APC9 Apc9 --- --- ---

APC10 Apc10/Doc1 Apc10 Apc10 DmApc10

APC11 Apc11 Apc11 Apc11 lemming

APC12 Cdc26 Hcn1 Cdc26 Cdc26

APC13 Swm1 Apc13 Apc13 Apc13

The APC subunits are evolutionarily conserved

A speculative model of protein ubiquitylation by the APC

What about the protease?

• Previous studies demonstrated that the activity of the protease was ATP dependent (not just ubiquitination requires ATP)

• What is it composed of?• Where is it located?• How is it selective toward ubiquitinated

proteins?• Why does it need ATP?

Proteasomethe cellular chamber of doom

Composed of at least 64 subunits with a molecular mass of about 2.5 MDa

Barrel-shaped 20S catalytic core particle

Two 19S regulatory cap particles

Major substrates: polyubiquitylated proteins

Cleaves proteins in an ATP dependent manner

Proteasome structure

The structure and subunit composition of proteasomes purified from different species or by different protocols are almost identical.

The Core Particle (CP)- made of 2 copies of each of 14 different proteins. - these are assembled in groups of 7 forming a ring. - the 4 rings are stacked on each other (like 4 doughnuts).

The Regulatory Particle (RP)- two identical RPs, one at each end of the core particle. - each is made of 18 different proteins - 6 of these are ATPases. - some of the subunits recognize the protein ubiquitin.

Mechanism of protein degradation

- The complex binds to ubiquitin-recognizing site(s) on the regulatory particle.

- The protein is unfolded by the ATPases using the energy of ATP.

- The unfolded protein is translocated into the central cavity of the core particle.

- Several active sites on the inner surface of the two middle "doughnuts" break various specific peptide bonds of the chain.

- This produces a set of peptides averaging about 8 amino acids long. These leave the core particle

- The regulatory particle releases the ubiquitins for reuse.

The active sites of the proteasome

Proteasomes degrade proteins in a highly processive fashion

"bite-chew" model

active sites with chymotrypsin, trypsin and caspase-like activities

active sites work in an organized manner

active sites regulate each other's activity

products are small oligopeptides with 3-20 residues

Since many substrate proteins and manyprocesses are involved in ubiquitylation, it is notsurprising that malfunctions of the ubiquitin-proteasome system have been implicated –directly or indirectly – in the etiology of manyinherited and acquired human diseases.

Ubiquitin system and disease

Ubiquitylation is reversible

ProteinProtein

DUBsDeubiquitylating enzymes

E1, E2, E3Ubiquitylating enzymes

Deubiquitylating enzymes (DUBs)Deubiquitylating enzymes (DUBs)

Cysteine-proteasesMetallo-

proteases

Catalytic functions of DUBs

Processes regulated by ubiquitin-mediated protein degradation

ApoptosisCell cycle

Development

Stress response

Protein degradation

Antigen presentation

InflammationCancer

Neuronal abnormalities

Metabolism

transcription

DNA repair

Signaltransduction

Human pathologies resulting from disorders in protein degradation

Mohammed AliPope John Paul II Michael J. Fox

Parkinson disease

synuclein synuclein

parkin

parkin

Dopamine producingcells die

von Hippel-Lindau syndrome

• Caused by mutation of the VHL tumor suppressor gene.

• This leads to the development of angio-blastoma in the CNS and the retina.

• Also leads to the development of cysts in kidneys and renal cell carcinoma.

The VHL protein regulates the degradation of HIF transcription factor

HIFVHL

oxygen

hypoxia inducible factor

Mutation of the VHL gene leads to overproduction of growth factors

HIF HIF

VHL

VEGF TGF-αGrowth factors

Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.

Diverse biological roles of protein ubiquitination

Gabriella EndreInstitute of Genetics

October 25, 2017

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

Ubiquitin with purple Lysine Residues

Ubiquitin

Ubiquitous in nature (hence the name)

Small polypeptide of 76 AA

Highly conserved in evolution: 3 AA differences between yeast and human homologues

Can mono or poly-ubiquinate

Lys-48 linked poly-ubiquitin chains common - proteasome

1975 - molecule

Ubiquitin processing and conjugation

early 1980s – pathway

and Irwin Rose University of California, Irvine, USA

Nobel Prize in Chemistry 2004

Aaron Ciechanover, Avram HershkoTechnion – Israel Institute of Technology, Haifa, Israel

Ubiquitin conjugation to substrates

ATP+

E1

E1

Activating

E2

Conjugating Ligating

E2

E2RING E3

E2HECT E3

substrate

substrate

12

substrate

ActivatingE1

ConjugatingE2

LigatingE3

Ub: general term here for monoubiquitin any or polyubiquitin chain

UPSUbiquitin - Proteasome System

UMSUbiquitin - Modification System

Degradation of proteins in proteosome

Alteredlongevity localizationand/or activity

of proteins

Ubiquitin labelling is not always fatal for the protein!

several non-proteolytic functions associated with the addition of

- a single ubiquitin molecule (mono-ubiquitination)

- or specific cases of polyubiquitination

affecting the substrate’s

cellular sub-location,

function

or its degradation through lysosomes

Ub modifications with their functional roles

Fates of ubiquitinated proteins

A: Monoubiquitinated integral membrane proteins are internalizedB: Polyubiquitinated integral membrane proteins can be degraded either by vacuoles or, via ERAD, the proteasomeC: Polyubiquitinated soluble proteins are proteasomally degraded (Lys-48) or play roles in DNA repair (Lys-63) D: Monoubiquitination and multiubiquitination of soluble proteins can lead to activation or inhibition of a protein’s activity

Guerra & Callis 2012 Plant Physiol160: 56–64

Example for the versatility of ubiquitination

I.

Regulation of Transcription Factors by ubiquitination

Regulating Transcription Factors (TFs) by ubiquitination

Three strategiesby controlling the

of the transcription factors

localization

activity

abundance

Regulating TFs by ubiquitinationlocalization

TF can be kept

outside the

nucleus by

interactions with

an inhibitor that

can be destroyed

by the UPS upon

a signal (like TF

NFκB and its

inhibitor IκB)

Another Ub-

family member

SUMO (S) can

directly

conjugate to

activators and

sequester them

into nuclear

bodies

I

Can regulate the association of activators with co-activator proteins

Regulating TFs by ubiquitination

either directly:

by blocking the

association of an

activator with its

essential cofactor

or indirectly:

by facilitating

the exchange

of cofactors

with

an activator

activity

abundance

I - constitutive turnovermaintaining an activator in a constitutively unstable form -prompt transcriptional response when appropriate signal comes

II - trx-coupled destructionactivators are destroyed during the act of transcriptional activation as a way of limiting uncontrolled activation by any one DNA bound TF

Regulating TFs by ubiquitination

Numerous effects of ubiquitinationin one TF pathway: NF-kB

identified E3 ligases

identified DUBs

Wertz and Dixit, 2010

Mechanisms for Modulating Substrate Recognition by E3s

Pickart, 2004 Cell 116: 181–190,

posttranslational modificationsand other mechanisms known to regulate the recognition of cognate substrates by different E3s.

Wertz and Dixit, 2010

Example of the numerous effects of ubiquitination in one TF pathway

Lys-48-linked ubiquitin chains – redLys-63-linked ubiquitin chains - green

Example for the versatility of ubiquitination

II.

UbK63 Modification of Endocytic Cargoes

Examples of trafficking steps that involve UbK63 chains

Several channels, transporters and receptors undergo modification by UbK63 chains These endocytic cargoes are recognized for sorting to invaginated regions of the plasma membrane by a number of UBD-containing proteins.

Erpapazoglou et al., 2014 Cells

Example for the versatility of ubiquitination

III.

K63-Linked Ubiquitination in Selective Autophagy

Erpapazoglou et al., 2014 Cells

UbK63 chains positively regulate the autophagic clearance of aggresomes, mitochondria andintracellular bacteria by interfering with various steps of the process.

Involvement of UbK63 chains in selective autophagy

HOW the versatility of ubiquitinationis achieved?

Modulating Substrate Recognition by E3s

Mechanisms for Modulating Substrate Recognition by E3s

Pickart & Eddins 2004 BBA 1695: 55–72

Budding yeast

1 – E1

11 – E2

42 – E3U-box subclass: 2

>>E2/E3combinations

Plant Arabidopsis

2– E1

34– E2

>1300 – E3U-box subclass: 64

>>>>E2/E3combinations

Variety of domain compositions and organization for plant U-box proteins

Yee & Goring 2009 J. Exp. Botany

Many examples for the versatility of ubiquitination

in plants

hormone signalling

tailoring morphogenesis

responses to environmental challenges

self recognition (pollination)

battling pathogens

…

Roles of specific E3s in hormone signalling and photoperiod measurement

Vierstra, 2009 Nature Reviews

Control of self-incompatibility in flowers by the ubiquitin–26S proteasome system

Vierstra, 2009 Nature Reviews

Scheuring et al., 2009 BMC Plant Biol.

Sorting of plasma membrane and Golgi proteins into the vacuolar

degradation pathway

Model illustrating ubiquitin-mediated vacuolar transport of membrane proteins

Scheuring et al., 2009 BMC Plant Biol.

Ubiquitin causes internalization of a non-secretory reporter at the PM

Expression of non-secretory reporters to analyze the endocytic pathway.

Scheuring et al., 2009 BMC Plant Biol.

Ubiquitin causes a plasma membrane protein to traffic to the vacuole

Ubiquitin-dependent transport of an integral PM protein to the vacuole.

Scheuring et al., 2009 BMC Plant Biol.

Ubiquitin directs Golgi-localized proteins to the vacuole

Ubiquitin-dependent transport from the Golgi to the vacuole.

Ubiquitin ligase proteins during the regulation of plant immune signaling

Duplan & Rivas, 2014 Frontiers in Plant Science

Microbial effector proteins acting as or interacting with E3 ligase proteins in the host.

Examples of positive and negative regulatory roles on plant immunity by host E3 Ub-ligases.

Microbial effector proteins acting as ubiquitin E3 ligases???

YES!

Pathogens have “learned” how to (try to) fool the host

Some pathogen effectors interferingwith the plant ubiquitin system

Marino et al., 2012 Plant Physiol.

U-box and F-box effector proteins interfering directly or indirectly with the host UPS are color coded according to the pathogenicorganism and respectively representedby U and F symbols.

Control of Agrobacterium infection by VirF

Vierstra, 2009 Nature Reviews

During the pathogenesis cycle, a single-stranded DNA (T-strand) is synthesized from the Ti plasmid, coated withvirulence protein-E2 (VirE2) and transported into the plant host along with the VirF protein through the type-3secretion system (T3SS)

Microbial effector proteins acting as ubiquitin E3 ligases???

YES!

Pathogens have “learned” how to (try to) fool the host

Not only with plants, butGeneral feature

Manipulation of host ubiquitin pathwaysby Salmonella

Perrett et al., 2011 Frontiers in Microbiol.

Manipulation of host ubiquitin pathwaysby Yersinia

Perrett et al., 2011 Frontiers in Microbiol.

UBIQUITINATION

A VERSATILE POSTTRANSLATIONAL MODIFICATION

Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.