Gold for Ubiquitin in Vancouver

The rise of proteomics has had tremendous influence on analysis and understanding of the role of post-translational modifications in biological processes. The covalent attachment of small proteins like ubiquitin, SUMO, 1The abbreviations used are:SUMOsmall ubiquitin-like modifierUblubiquitin-like proteinUPSubiquitin-proteasome systemVCPvalosin-containing proteinIBMPFDinclusion body myopathy associated with Paget disease of bone and frontotemporal dementiaSILACstable isotope labeling with amino acids in cell cultureDUBdeubiquitinating enzymeCRLCullin-RING ligaseCSNCOP9 signalosome complexAQUAabsolute quantitation by mass spectrometrySCFSkp1, Cullin, F-box. or other ubiquitin-like proteins (Ubls) is one class of post-translational modifications where proteomics has had notable impact. Various proteomics approaches, but in particular mass spectrometry-based analyses, have influenced the field and enabled significant advances over the past few years. The first meeting dedicated to proteomics of protein degradation and ubiquitin pathways showcased these advances and allowed a glimpse at future contributions of proteomics to this field. With its many attractive drug targets, the ubiquitin and proteasome system, as well as other proteolysis pathways, could offer new therapies for various human diseases including cancer and neurodegenerative disorders.The covalent linkage of ubiquitin to other proteins is catalyzed by the E1-E2-E3 cascade of enzymatic reactions whereby the many different E3 ubiquitin ligases provide substrate specificity to the process of protein ubiquitylation (1Pickart C.M. Back to the future with ubiquitin.Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Ubiquitylation is best known for targeting proteins for degradation by the proteasome, but other functions for ubiquitylation independent of proteolysis are also known. Likewise, modifications with SUMO or other Ubls generally do not regulate protein degradation but instead control subcellular localization, protein interactions, or change protein conformation and activity (2Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (602) Google Scholar).The questions addressed by proteomics approaches to ubiquitylation and Ubl modifications are plentiful. They range from very specific, e.g. determination of the modified residue in a substrate protein, to complex, such as protein dynamics in proteome-wide ubiquitin (or Ubl) modification profiles (3Kirkpatrick D.S. Denison C. Gygi S.P. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics.Nat. Cell Biol. 2005; 7: 750-757Crossref PubMed Scopus (189) Google Scholar). In either case, the rapid technological advancements (particularly in mass spectrometry instrumentation as well as quantitation and separation technologies) have allowed impressive progress, which was evident in the First Conference on Proteomics of Protein Degradation and Ubiquitin Pathways in Vancouver (http://ppdup.org/) (Fig. 1). The rise of proteomics has had tremendous influence on analysis and understanding of the role of post-translational modifications in biological processes. The covalent attachment of small proteins like ubiquitin, SUMO, 1The abbreviations used are:SUMOsmall ubiquitin-like modifierUblubiquitin-like proteinUPSubiquitin-proteasome systemVCPvalosin-containing proteinIBMPFDinclusion body myopathy associated with Paget disease of bone and frontotemporal dementiaSILACstable isotope labeling with amino acids in cell cultureDUBdeubiquitinating enzymeCRLCullin-RING ligaseCSNCOP9 signalosome complexAQUAabsolute quantitation by mass spectrometrySCFSkp1, Cullin, F-box. or other ubiquitin-like proteins (Ubls) is one class of post-translational modifications where proteomics has had notable impact. Various proteomics approaches, but in particular mass spectrometry-based analyses, have influenced the field and enabled significant advances over the past few years. The first meeting dedicated to proteomics of protein degradation and ubiquitin pathways showcased these advances and allowed a glimpse at future contributions of proteomics to this field. With its many attractive drug targets, the ubiquitin and proteasome system, as well as other proteolysis pathways, could offer new therapies for various human diseases including cancer and neurodegenerative disorders. small ubiquitin-like modifier ubiquitin-like protein ubiquitin-proteasome system valosin-containing protein inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia stable isotope labeling with amino acids in cell culture deubiquitinating enzyme Cullin-RING ligase COP9 signalosome complex absolute quantitation by mass spectrometry Skp1, Cullin, F-box. The covalent linkage of ubiquitin to other proteins is catalyzed by the E1-E2-E3 cascade of enzymatic reactions whereby the many different E3 ubiquitin ligases provide substrate specificity to the process of protein ubiquitylation (1Pickart C.M. Back to the future with ubiquitin.Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). Ubiquitylation is best known for targeting proteins for degradation by the proteasome, but other functions for ubiquitylation independent of proteolysis are also known. Likewise, modifications with SUMO or other Ubls generally do not regulate protein degradation but instead control subcellular localization, protein interactions, or change protein conformation and activity (2Hochstrasser M. Origin and function of ubiquitin-like proteins.Nature. 2009; 458: 422-429Crossref PubMed Scopus (602) Google Scholar). The questions addressed by proteomics approaches to ubiquitylation and Ubl modifications are plentiful. They range from very specific, e.g. determination of the modified residue in a substrate protein, to complex, such as protein dynamics in proteome-wide ubiquitin (or Ubl) modification profiles (3Kirkpatrick D.S. Denison C. Gygi S.P. Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics.Nat. Cell Biol. 2005; 7: 750-757Crossref PubMed Scopus (189) Google Scholar). In either case, the rapid technological advancements (particularly in mass spectrometry instrumentation as well as quantitation and separation technologies) have allowed impressive progress, which was evident in the First Conference on Proteomics of Protein Degradation and Ubiquitin Pathways in Vancouver (http://ppdup.org/) (Fig. 1). The two plenary lectures were delivered by Ray Deshaies (Pasadena, CA) and Dan Finley (Boston, MA), who were among the first to exploit mass spectrometry-based proteomics to address mechanistic questions of the ubiquitin-proteasome system (UPS). R. Deshaies presented work that analyzed the role of the AAA-ATPase Cdc48/p97/VCP in protein degradation (4Jentsch S. Rumpf S. Cdc48 (p97): a “molecular gearbox” in the ubiquitin pathway?.Trends Biochem. Sci. 2007; 32: 6-11Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The yeast Cdc48 and its highly conserved mammalian homolog p97/VCP have long been connected with the UPS and are thought to deliver ubiquitylated proteins to the proteasome for degradation. Consistent with this role in proteasome targeting, the Deshaies group noticed that in yeast mutants carrying a temperature-sensitive allele of CDC48 or after p97 knockdown in mammalian cells levels of total ubiquitylated proteins are significantly increased. However, when proteasomes were purified from yeast cells, they found more ubiquitin chains bound to the proteasome in cdc48 mutants than in wild-type cells. This surprising result argues against the simple model for Cdc48 in substrate delivery to the proteasome, which predicted reduced levels of ubiquitin conjugates at the proteasome after Cdc48 inactivation. R. Deshaies then showed mass spectrometric analyses comparing proteasome-interacting proteins in wild-type cells and cdc48 mutants and found that many proteasome-interacting proteins were specific for cdc48 mutants, suggesting the intriguing possibility that degradation substrates accumulate at the proteasome in cells lacking Cdc48. One of these proteins, the RNA polymerase II subunit Rpb1, was analyzed in more detail. Rpb1 is ubiquitylated and degraded in response to UV radiation. cdc48 mutants accumulated ubiquitylated Rpb1 after UV radiation, and significant amounts were bound to proteasomes. These findings suggest that for Rpb1 the primary role for Cdc48 function appears to be late in the degradation process after substrate arrives at the proteasome rather than in an earlier targeting step. R. Deshaies also reported system-wide quantitative ubiquitin profiling experiments that compared ubiquitin profiles in wild-type and cdc48 mutants. They found that the levels of about 10% of ubiquitylated proteins were increased in cells lacking Cdc48 activity, further underscoring the importance of Cdc48 for the UPS. Several other presentations highlighted the growing interest in the diverse roles of the AAA-ATPase Cdc48/p97. For example, Peter Kaiser (Irvine, CA) described a quantitative proteomics approach to understand how missense mutations in human p97/VCP cause the hereditary disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), a fatal form of inclusion body myopathy with no available cure. The SILAC-based MS strategy revealed dramatically increased binding of some p97 cofactors, in particular Npl4, to the disease-causing p97/VCP mutants. This gain of function behavior is consistent with the dominant character of IBMPFD and supports a recent report suggesting p97 cofactor imbalance as a cause for this human disease (5Fernández-Sáiz V. Buchberger A. Imbalances in p97 co-factor interactions in human proteinopathy.EMBO Rep. 2010; 11: 479-485Crossref PubMed Scopus (77) Google Scholar). D. Finley presented interesting work on a new inhibitor of the USP14/Ubp6 deubiquitinating enzyme (DUB), which is associated to the proteasome. Although DUB activity at the proteasome is required to allow the degradation of ubiquitylated substrates (and permit their entrance in the 20 S chamber), it may also prevent the degradation of substrates by removing ubiquitin before their targeting for proteolysis. The Finley team reasoned that inhibition of such DUB activity may accelerate the degradation of a pool of substrates like misfolded proteins. They identified, in collaboration with the group of Randy King (Boston, MA), a new reversible inhibitor targeting USP14 and found that it increased the degradation of model substrates like cyclinB and Sic1PYp by the proteasome. Remarkably, the compound reduced the levels of several aggregation-prone proteins (e.g. TPD43) in mouse embryonic fibroblast cells expressing USP14 but not in USP14−/− control cells. In addition, after oxidative damage, fewer oxidized proteins accumulated in the presence of the USP14 inhibitor, resulting in an increase of cell viability. This exciting work opens new possibilities in the field where proteasome-dependent degradation may be up-regulated by specific compounds. It also nicely illustrates how the proteasome itself plays an active role in regulating proteolysis by remodeling the ubiquitin chains of substrates. The role of DUBs in the ubiquitin system was further illustrated in other presentations. Notably, the laboratory of Kathy Gould (Nashville, TN) performed a “global census” of the 20 DUBs in fission yeast by combining analyses of localization, binding partners (using mass spectrometry), and mutation-associated phenotypes. They assigned several new functions for DUBs based on their census profile. For instance, Ubp4 and Ubp5 were found to function at the endosome and Golgi, respectively. Remarkably, DUBs displayed an astonishing capacity to compensate for other DUB gene deletion. For instance, no less than five DUB deletions were required to obtain a strong defect in endocytosis. Richard Gardner (Seattle, WA) presented results on Ubp10 in budding yeast where new substrates were identified after comparing data obtained from two different proteomics approaches. In the first approach, Ubp10 binding partners were identified after pulldown following in vivo cross-linking. In the second approach, proteins more ubiquitylated in ubp10Δ cells (due to the absence of the DUB) were identified after purifying ubiquitylated proteins and comparing them with wild-type cells. These experiments confirmed the role of Ubp10 in gene silencing and suggested a new role in 40 S ribosome biogenesis. E3 ubiquitin ligases are considered the key regulators of the UPS. Wade Harper (Boston, MA) presented a comprehensive proteomics approach to understand Cullin-RING ligases (CRLs) (6Petroski M.D. Deshaies R.J. Function and regulation of cullin-RING ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1692) Google Scholar). CRLs are multisubunit ligases assembled on a cullin scaffold. Generally, they consist of two to three components forming the core ligase involved in E2 recruitment and activation and a substrate adapter for substrate selection. Hundreds of substrate adapters can dock to a few different core ligases, generating the substantial diversity of this ligase family. These adapter interactions can be key nodes for E3 regulatory processes. Studies in fission yeast suggested that the COP9 signalosome complex CSN as well as the protein CAND1, both tightly linked to the cullin neddylation cycle, regulate adapter subunit binding at various levels (7Schmidt M.W. McQuary P.R. Wee S. Hofmann K. Wolf D.A. F-box-directed CRL complex assembly and regulation by the CSN and CAND1.Mol. Cell. 2009; 35: 586-597Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). W. Harper's team approached the dynamics of CRL regulation by constructing a quantitative proteomics platform to systematically examine CRL architecture as CRLs transition from active to inactive states using an inhibitor of the Nedd8 conjugation pathway (MLN4924). Utilizing a multiplex absolute quantitation by mass spectrometry (AQUA)-based quantitative MS approach, they were able to analyze not only the global composition of CRLs but also the absolute stoichiometry of the various CRL components. This approach can be broadly applied to examine other protein signaling networks and how their composition and post-translational modifications change in response to pathway perturbations. As an interesting technical note, W. Harper mentioned that a significant preservation of the neddylation status during purification is achieved by the metalloprotease inhibitor o-phenanthroline, which inhibits the zinc-dependent deneddylation activity of CSN. Poul Sorensen (Vancouver, British Columbia, Canada) reported an interesting connection between the tumor suppressor Hace1 and CAND1. Genetic experiments by the Sorensen team suggest that Hace1 functions as an inhibitor of several CRLs. Using mass spectrometry, they identified CAND1 as a major interactor of Hace1 and found that CAND1 binding was significantly increased under nutritional stress or hypoxia. Under these same conditions, less CAND1 was bound to cullins, implying that Hace1 acts by CAND1 sequestration. Millennium Pharmaceuticals has developed the neddylation inhibitor MLN4924 (used in the aforementioned work by W. Harper), which targets the Nedd8 activating enzyme and is in clinical trials in patients with advanced malignancies. Cullin modification by the ubiquitin-like protein Nedd8 is crucial for CRL activity, and MLN4924 thus functions indirectly as a potent CRL inhibitor (9Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. Cullis C.A. Doucette A. Garnsey J.J. Gaulin J.L. Gershman R.E. Lublinsky A.R. McDonald A. Mizutani H. Narayanan U. Olhava E.J. Peluso S. Rezaei M. Sintchak M.D. Talreja T. Thomas M.P. Traore T. Vyskocil S. Weatherhead G.S. Yu J. Zhang J. Dick L.R. Claiborne C.F. Rolfe M. Bolen J.B. Langston S.P. An inhibitor of NEDD8-activating enzyme as a new approach to treat 2009; 458: PubMed Scopus Google Scholar). of the and CRL substrate and of have been identified as the primary for cancer cell of MA) reported to and pathways for MLN4924 experiments were used to over proteins that were MLN4924 in cells. strategy was then used to the identified proteins based on the of their knockdown and MLN4924 on cell of or knockdown of the CRL adapter or E2 the of The by the of approaches that proteomics with profiling Ubiquitylation by CRLs has been thought to be at the of substrate but the dynamics of CRL is an that also offer of Kaiser reported active of the yeast CRL The Kaiser group showed that stress of the adapter subunit which Cdc48 that in from the core to ligase inactivation. quantitative mass spectrometric of ligase in yeast showed that was of adapter components as a for CRL Various presented results obtained from mass spectrometric of ubiquitin conjugates after Notably, the of R. 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Deshaies showed how quantitative mass spectrometry can be used to the and specificity of the by comparing proteins from cells, which do or do not the The diverse that have been developed for ubiquitin profiling be to the of ubiquitin ligase substrates by comparing ubiquitin profiles of wild-type cells with ligase mutants. the of ubiquitin ligases and the of substrate that can be associated with these ligases, such an approach have tremendous on and few for substrate have been suggesting that further of and instrumentation are (Vancouver, British Columbia, Canada) used ubiquitin and quantitative MS to that a rapid increase of ubiquitylation of proteins in budding which for a ubiquitin was found to be involved in this stress response comparing ubiquitylated proteins from and wild-type cells, group was able to proteins that were more ubiquitylated in wild-type cells, suggesting that they may be substrates. for ligase substrate have been reported by and reported that proteasome inhibition can be used to the complex, and mass spectrometric of ligase bound proteins can thus substrates. 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In with showed that the proteasome is very active in the degradation of substrates modified with ubiquitin is a growing that other ubiquitin such as as potent proteasome degradation and that both and are involved in The proteasome is a complex that has been by mass spectrometry in yeast and mammalian cells. presented proteomics analysis of the proteasome. by using a approach, the laboratory found that the 20 S subunit was like the other three suggesting that the proteasome may a of the proteasome has been by its as the of the cell that is active with of the regulation at the ubiquitylation more with many of proteasome and specific regulatory pathways is not the to proteomics approaches. 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J. an attractive model whereby a of activity can be by the proteasome, to activation the proteasome and to (Vancouver, British Columbia, Canada) presented an of proteomics that group developed to For instance, by using an approach to amino they were able to for by and to analyze them by mass With over in the human be no of work for in understanding of the ubiquitin and ubiquitin-like system has in the past several years. It has also with over human involved in ubiquitylation and Ubl the system is are a to the of the One is to the and the functions of the different ubiquitin and Ubl and analysis for have to a of new data in recent the of several of For only a few of ubiquitylation have been identified to new proteomics approaches to further in the ubiquitin field. With for different ubiquitin in vivo as well as the possibility of the roles of these different to be and to substrates modified with different chains are thus required to biological In addition, comprehensive to which substrates are by the different E3 ligases to be Hofmann presented some and analyses to with several of the in the field. proteomics analysis of E3 ligases, as performed by the Harper provide a of new major in the field be to and the and new results in or new One of the Proteomics of Protein Degradation and Ubiquitin was to major key in the proteomics and ubiquitin field to these different are to this in the the for to their presentations and to work could not be of also the for an interesting and of British for on the

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