Ubiquitylation: its role and medical significance

Transcription

1 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Ubiquitylation: its role and medical significance Zoltán Lipinszki Institute of Biochemistry October 25, 2017

2 Cellular homeostasis 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.

3 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

4 Schoenheimer: proteins are in a dynamic turnover

5 De Duve: protein degradation in lysosomes

6 Energy dependence of protein degradation tyrosine aminotransferase

7 Az ATP-függő proteolitikus aktivitás frakcionálása DEAE-cellulóz kromatográfiával TABLE 1.- Resolution of the ATP-dependent cell-free proteolytic system into complementing activities

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9 Nobel Prize in Chemistry, 2004 Aaron Ciechanover Avram Hershko Irwin Rose "for the discovery of ubiquitin-mediated protein degradation"

10 Discovery timeline of the ubiquitin proteasome system

11 Cyclin levels fluctuate during cell cycle

12 Mitotic cyclin destruction box H 2 N COOH Cyclin A Cyclin B1 Cyclin 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

13 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

14 The structure of ubiquitin is conserved from yeast to human 1-MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF-45 1-MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG AGKQLEDGRTLADYNIQKESTLHLVLRLRGG AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG-76 Fission yeast Green pea fruitfly human

15 Ubiquitin is synthesized as a polyprotein Ubiquitin Ribosomal protein Ubiquitin Ubiquitin Ubiquitin Transcription/Translation Ub C-terminal hydrolase

16 Ubiquitin is conjugated to proteins

17 Enzymatic cascade of protein ubiquitylation O C OH Ub ATP AMP Ub: ubiquitin E1: ubiquitin-activating enzyme E2: ubiquitin-conjugating enzyme E3: ubiquitin ligase O C S Ub E1 O C S Ub E2 Degradation by the 26S proteasome E3 O C NH Ub Ub Ub Ub S

18 The ubiquitin system is enormous The genes of the UPS constitutes ~5% of the genome E1 s activating enzymes E2 s conjugating enzymes E3 s ubiquitin ligase - drives specificity DUBs ubiquitin specific proteases

19 The hierarchical structure of the ubiquitin system

20 The APC subunits are evolutionarily conserved 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 APC Apc7 DmApc7 APC8 Cdc23 Cut23 Apc8/Cdc23 DmApc8 APC9 Apc APC10 Apc10/Doc1 Apc10 Apc10 DmApc10 APC11 Apc11 Apc11 Apc11 lemming APC12 Cdc26 Hcn1 Cdc26 Cdc26 APC13 Swm1 Apc13 Apc13 Apc13

21 A speculative model of protein ubiquitylation by the APC

22 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?

23 Proteasome the 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

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

25 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.

26 The active sites of the proteasome

27 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

28 Ubiquitin system and disease Since many substrate proteins and many processes are involved in ubiquitylation, it is not surprising that malfunctions of the ubiquitinproteasome system have been implicated directly or indirectly in the etiology of many inherited and acquired human diseases.

29 Ubiquitylation is reversible Ubiquitylating enzymes E1, E2, E3 Protein Protein Deubiquitylating enzymes DUBs

30 Deubiquitylating enzymes (DUBs) Cysteine-proteases Metalloproteases

31 Catalytic functions of DUBs

32 Processes regulated by ubiquitinmediated protein degradation DNA repair transcription Metabolism Signal transduction Cell cycle Neuronal abnormalities Cancer Apoptosis Development Stress response Protein degradation Antigen presentation Inflammation

33 Human pathologies resulting from disorders in protein degradation Pope John Paul II Michael J. Fox Mohammed Ali

34 Parkinson disease synuclein synuclein parkin parkin Dopamine producing cells die

35 von Hippel-Lindau syndrome Caused by mutation of the VHL tumor suppressor gene. This leads to the development of angioblastoma in the CNS and the retina. Also leads to the development of cysts in kidneys and renal cell carcinoma.

36 The VHL protein regulates the degradation of HIF transcription factor HIF VHL hypoxia inducible factor oxygen

37 Mutation of the VHL gene leads to overproduction of growth factors HIF VHL HIF VEGF Growth factors TGF-α

38 Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project.

39 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Diverse biological roles of protein ubiquitination Gabriella Endre Institute of Genetics October 25, 2017

40 Ubiquitin Ubiquitous in nature (hence the name) molecule 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 Ubiquitin with purple Lysine Residues

41 Ubiquitin processing and conjugation early 1980s pathway Nobel Prize in Chemistry 2004 Aaron Ciechanover, Avram Hershko Technion Israel Institute of Technology, Haifa, Israel and Irwin Rose University of California, Irvine, USA

42 Ubiquitin conjugation to substrates E2 RING E3 substrate E1 E2 ATP+ Activating E1 Conjugating E2 Ligating 1 E2 2 HECT E3 substrate substrate Activating E1 Conjugating E2 Ligating E3 Ub: general term here for monoubiquitin any or polyubiquitin chain

43 UPS Ubiquitin - Proteasome System Degradation of proteins in proteosome UMS Ubiquitin - Modification System Altered longevity localization and/or activity of proteins

44 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

45 Ub modifications with their functional roles

46 Fates of ubiquitinated proteins Guerra & Callis 2012 Plant Physiol160: A: Monoubiquitinated integral membrane proteins are internalized B: Polyubiquitinated integral membrane proteins can be degraded either by vacuoles or, via ERAD, the proteasome C: 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

47 Example for the versatility of ubiquitination I. Regulation of Transcription Factors by ubiquitination

48 Regulating Transcription Factors (TFs) by ubiquitination Three strategies by controlling the localization activity abundance of the transcription factors

49 Regulating TFs by ubiquitination TF can be kept outside the nucleus by interactions with an inhibitor that I can be destroyed by the UPS upon a signal (like TF NFκB and its inhibitor IκB) localization Another Ubfamily member SUMO (S) can directly conjugate to activators and sequester them into nuclear bodies

50 Regulating TFs by ubiquitination activity Can regulate the association of activators with co-activator proteins either directly: by blocking the association of an activator with its essential cofactor or indirectly: by facilitating the exchange of cofactors with an activator

51 Regulating TFs by ubiquitination abundance I - constitutive turnover maintaining an activator in a constitutively unstable form - prompt transcriptional response when appropriate signal comes II - trx-coupled destruction activators are destroyed during the act of transcriptional activation as a way of limiting uncontrolled activation by any one DNA bound TF

52 Numerous effects of ubiquitination in one TF pathway: NF-kB identified E3 ligases identified DUBs Wertz and Dixit, 2010

53 Mechanisms for Modulating Substrate Recognition by E3s posttranslational modifications and other mechanisms known to regulate the recognition of cognate substrates by different E3s. Pickart, 2004 Cell 116: ,

54 Example of the numerous effects of ubiquitination in one TF pathway Lys-48-linked ubiquitin chains red Lys-63-linked ubiquitin chains - green Wertz and Dixit, 2010

55 Example for the versatility of ubiquitination II. UbK63 Modification of Endocytic Cargoes

56 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

57 Example for the versatility of ubiquitination III. K63-Linked Ubiquitination in Selective Autophagy

58 Involvement of UbK63 chains in selective autophagy UbK63 chains positively regulate the autophagic clearance of aggresomes, mitochondria and intracellular bacteria by interfering with various steps of the process. Erpapazoglou et al., 2014 Cells

59 HOW the versatility of ubiquitination is achieved? Modulating Substrate Recognition by E3s

60 Budding yeast Mechanisms for Modulating Substrate Recognition by E3s Plant Arabidopsis 1 E1 2 E1 11 E2 34 E2 42 E3 U-box subclass: 2 >>E2/E3 combinations Pickart & Eddins 2004 BBA 1695: >1300 E3 U-box subclass: 64 >>>>E2/E3 combinations

61 Variety of domain compositions and organization for plant U-box proteins Yee & Goring 2009 J. Exp. Botany

62 Many examples for the versatility of ubiquitination in plants hormone signalling tailoring morphogenesis responses to environmental challenges self recognition (pollination) battling pathogens

63 Roles of specific E3s in hormone signalling and photoperiod measurement Vierstra, 2009 Nature Reviews

64 Control of self-incompatibility in flowers by the ubiquitin 26S proteasome system Vierstra, 2009 Nature Reviews

65 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.

66 Ubiquitin causes internalization of a non-secretory reporter at the PM Expression of nonsecretory reporters to analyze the endocytic pathway. Scheuring et al., 2009 BMC Plant Biol.

67 Ubiquitin causes a plasma membrane protein to traffic to the vacuole Ubiquitindependent transport of an integral PM protein to the vacuole. Scheuring et al., 2009 BMC Plant Biol.

68 Ubiquitin directs Golgi-localized proteins to the vacuole Ubiquitindependent transport from the Golgi to the vacuole. Scheuring et al., 2009 BMC Plant Biol.

69 Ubiquitin ligase proteins during the regulation of plant immune signaling Examples of positive and negative regulatory roles on plant immunity by host E3 Ubligases. Microbial effector proteins acting as or interacting with E3 ligase proteins in the host. Duplan & Rivas, 2014 Frontiers in Plant Science

70 Microbial effector proteins acting as ubiquitin E3 ligases??? YES! Pathogens have learned how to (try to) fool the host

71 Some pathogen effectors interfering with the plant ubiquitin system U-box and F-box effector proteins interfering directly or indirectly with the host UPS are color coded according to the pathogenic organism and respectively represented by U and F symbols. Marino et al., 2012 Plant Physiol.

72 Control of Agrobacterium infection by VirF During the pathogenesis cycle, a single-stranded DNA (T-strand) is synthesized from the Ti plasmid, coated with virulence protein-e2 (VirE2) and transported into the plant host along with the VirF protein through the type-3 secretion system (T3SS) Vierstra, 2009 Nature Reviews

73 Microbial effector proteins acting as ubiquitin E3 ligases??? YES! Pathogens have learned how to (try to) fool the host Not only with plants, but General feature

74 Manipulation of host ubiquitin pathways by Salmonella Perrett et al., 2011 Frontiers in Microbiol.

75 Manipulation of host ubiquitin pathways by Yersinia Perrett et al., 2011 Frontiers in Microbiol.

76 UBIQUITINATION A VERSATILE POSTTRANSLATIONAL MODIFICATION

77 Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project.