Proteomics Propels Protein Degradation Studies in San Diego

Exquisite in vitro biochemical examinations of protein ubiquitylation and degradation have historically been the dominant methods for unraveling the mechanisms of protein destruction. The study of protein abundance alterations and protein modifications, a cornerstone of protein degradation pathways, naturally lends itself to global and systematic proteomic methods to decipher the emerging complexity of protein degradation pathways. Advances in proteomic technologies have fueled an explosion of systematic and quantitative studies aimed at understanding how the proteome is shaped and regulated by ubiquitin-dependent processes. These types of studies, as well as targeted analyses of cellular pathways, have revealed that alterations in protein degradation function can have a severe impact on human health and disease. The fusion of these two themes was the focus of the January 2012 conference on proteomics of protein degradation and ubiquitin pathways (PPDUP) held in San Diego. To gain insights into both the current state-of-the-art proteomic methods to investigate protein turnover, and how protein degradation function is altered within a range of human disorders a variety of speakers revealed the many connections between altered protein degradation function and human disease. Many of the sessions were framed by a consistent focus aimed at the discovery and development of novel therapeutics targeting protein degradation pathway components to treat various human maladies ranging from cancer to heart disease. Exquisite in vitro biochemical examinations of protein ubiquitylation and degradation have historically been the dominant methods for unraveling the mechanisms of protein destruction. The study of protein abundance alterations and protein modifications, a cornerstone of protein degradation pathways, naturally lends itself to global and systematic proteomic methods to decipher the emerging complexity of protein degradation pathways. Advances in proteomic technologies have fueled an explosion of systematic and quantitative studies aimed at understanding how the proteome is shaped and regulated by ubiquitin-dependent processes. These types of studies, as well as targeted analyses of cellular pathways, have revealed that alterations in protein degradation function can have a severe impact on human health and disease. The fusion of these two themes was the focus of the January 2012 conference on proteomics of protein degradation and ubiquitin pathways (PPDUP) held in San Diego. To gain insights into both the current state-of-the-art proteomic methods to investigate protein turnover, and how protein degradation function is altered within a range of human disorders a variety of speakers revealed the many connections between altered protein degradation function and human disease. Many of the sessions were framed by a consistent focus aimed at the discovery and development of novel therapeutics targeting protein degradation pathway components to treat various human maladies ranging from cancer to heart disease. Participants of the second Proteomic in Protein Degradation and Ubiquitin Pathways (PPDUP) 1The abbreviations used are:PTMpost translational modificationCRLcullin-RING ligasediGlydiGlycineDUBdeubiquitylating enzymePPDUPProteomics of Protein Degradation and Ubiquitin PathwaysROSreactive oxygen speciesTMTtandem-mass tagsUBLubiquitin-likeUPSubiquitin proteasome system. 1The abbreviations used are:PTMpost translational modificationCRLcullin-RING ligasediGlydiGlycineDUBdeubiquitylating enzymePPDUPProteomics of Protein Degradation and Ubiquitin PathwaysROSreactive oxygen speciesTMTtandem-mass tagsUBLubiquitin-likeUPSubiquitin proteasome system. conference, most of whom were escaping winter, met near San Diego, California to share their viewpoints and new results in the field. Keynote speakers Alfred Goldberg (Harvard Medical School, MA) and Mike Tyers (University of Montreal, Canada) opened the meeting and provided a comprehensive tour of the various biochemical activates associated with ubiquitin-dependent protein turnover. Goldberg presented an update to their work unraveling the biochemical processes associated with the proteasome to facilitate protein degradation, and Tyers reported on their efforts to characterize and pharmacologically inhibit ubiquitin conjugating enzymes. These early talks, together with Ralph Bradshaw (UCSF, CA) and Al Burlingame (UCSF, CA), provided insightful perspectives on previous work studying protein degradation pathways and their links to human disease. The conference was bookended by two closing keynote addresses from Ben Cravatt (Scripps Research Institute, CA) and Wade Harper (Harvard Medical School) who together demonstrated the power of proteomic methodologies to characterize and identify substrates for proteases and various ubiquitin-related enzymes as well as identify new roles for the complex array of enzymes that mediate ubiquitylation of proteins. These talks highlighted several important themes and topics that resonated throughout many of the talks providing a future prospectus on the use of proteomics toward more systematic investigations of protein modifications and their link to protein turnover and human health and disease. post translational modification cullin-RING ligase diGlycine deubiquitylating enzyme Proteomics of Protein Degradation and Ubiquitin Pathways reactive oxygen species tandem-mass tags ubiquitin-like ubiquitin proteasome system. post translational modification cullin-RING ligase diGlycine deubiquitylating enzyme Proteomics of Protein Degradation and Ubiquitin Pathways reactive oxygen species tandem-mass tags ubiquitin-like ubiquitin proteasome system. One of the largest challenges in the field of ubiquitin-dependent regulation has been the mapping and characterization of endogenous protein ubiquitylation and the identification of the exact lysine residues modified by ubiquitin. This is mainly because of the low abundance and stoichiometry of the fraction of the proteome that is ubiquitylated at any given time. However, studies from various labs over the past year have traversed this barrier through the use of antibody-based peptide enrichment strategies that use an antibody that specifically recognizes the diGlycine (diGly) remnant of ubiquitin that remains linked to modified lysine residues after trypsinolysis (1Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. A proteomics approach to understanding protein ubiquitination.Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1307) Google Scholar). Steve Gygi (Harvard Medical School, MA), in collaboration with Wade Harper's group (Harvard Medical School, MA) and Cell Signaling Technologies (CST, Danvers, MA) detailed their studies using a monoclonal diGly-specific antibody to enrich for modified peptides followed by quantification and identification by mass spectrometry. They reported the identification of over 17,000 diGly-modified lysine residues spread over ∼5000 proteins and characterized the alterations in abundance of many of these sites in response to proteasome inhibition. Using the same approach, Don Kirkpatrick (Genentech, CA) reported the identification of more than 4500 unique ubiquitin-modified peptides from mouse brain homogenates. Lastly, using an independent diGly-specific monoclonal antibody, Chunaram Choudhary (University of Copenhagen, Denmark) reported the identification of greater than 11,000 diGly-modified sites from tissue culture studies. Altogether, these studies illustrated a powerful new approach in which thousands of ubiquitin-modified lysines can be examined in a single experiment. This represents a great expansion in the scale of experiments that can be accomplished toward more global analyses of protein degradation. One of the caveats in using this strategy to capture potentially ubiquitylated peptides is that, because of the similarity in their C-terminal sequence, proteins that are modified by the ubiquitin-like proteins NEDD8 or ISG15 will leave identical diGly-remnants attached to lysines following trypsin digestion making the unequivocal identification of ubiquitylated proteins difficult. Gygi and colleagues extended their analysis to carefully measure the impact of other ubiquitin-like (UBL) modifications and estimate that over 94% of the identified sites likely are the result of ubiquitylation versus other UBL modifications. Choudhary presented a comparison of these diGly modified lysine residues to those identified to be acetylated revealing that diGly-modified lysine residues are more likely to be acetylated compared with unmodified lysines in the proteome; a finding that was also reported from the Gygi and Harper study. This suggests potential cross-talk between these two lysine post-translational modifications (PTMs) although careful studies interrogating individual lysine residues are needed to understand the functional significance of this overlap. Choudhary also reported preliminary studies comparing two distinct diGly-specific antibodies suggesting that, although they identify largely overlapping sets of modified peptides using the two different antibodies, there appeared to be a slight difference in the amino acid sequence surrounding the modified lysine residue recognized by either antibody. However, it should be noted that neither study was able to identify a specific ubiquitylation motif sequence, even with the large increase in the number of identified ubiquitin-modified peptides, suggesting a large plasticity in site-utilization by individual ubiquitin ligases. Using these new data sets of known sites of ubiquitin modification, Yaakov Levy (Weizmann Institute, Israel) took a bioinformatics approach to study the structural and evolutionary nature of ubiquitylated proteins and lysines. Levy reported that, surprisingly, ubiquitylated lysines were more often found in structured regions of the proteins and 38% of diGly-modified proteins do not contain an unstructured region within their entire coding sequence. However, because the used data sets contain experiments in which proteasome inhibitors were used it is uncertain if the identified proteins represent the fully folded and native portion of the proteome and caution should be taken when analyzing the structural characteristics of modified sequences. Indeed, studies from the Gygi and Harper groups suggest that a large fraction of the ubiquitin-modified proteome arises from newly translated proteins that may have not reached their final folded native state. Further, Levy reported that ubiquitin-modified lysines were more evolutionarily conserved compared with all lysines in the human proteome suggesting a possible selective pressure to maintain protein ubiquitylation over evolution. As an alternative to peptide-based enrichment methods, Manuel Rodriguez (Bizkaia, Spain) reported on the use of tandem repeated ubiquitin binding entities (TUBES) to isolate ubiquitin-modified proteins prior to digestion with trypsin. Rodriguez and colleagues have further developed this strategy to capture and identify sumoylated (another UBL modification) proteins as well, an often difficult task because of the lack of peptide enrichment methods for sumo-modified proteins. These studies set the stage for analysis of in vivo protein ubiquitylation in both mouse models of human disease as well as from human patient tissues to uncover new links between altered protein degradation and the onset of a myriad of human disorders. Another large, and often daunting, challenge to the field is the identification of substrates for ubiquitin ligases and deubiquitylating enzymes (DUBs) known to have roles in critical cellular pathways. As many cellular pathways are regulated by the precise degradation of components within crucial signaling hubs, identification of ubiquitin pathway components that govern this regulation would reveal potential candidates for therapeutic intervention. Use of the diGly-specific antibodies allows for the detection of the endogenously modified proteins which provide an opportunity to directly quantify the exact population of the proteome regulated by ligases and DUBs. Gygi and colleagues presented data using the diGly-capture approach to identify substrates for a family of multicomponent ubiquitin ligases known as cullin-RING ligases (CRL). Using a pharmacological inhibitor of the NEDD8 E1 enzyme, MLN4924, to specifically ablate the ubiquitylation of CRL targets, they reported the identification of more than 200 novel CRL substrates. Cellular apoptosis is another critical signaling pathway known to be regulated by the ubiquitin proteasome system. Don Kirkpatrick, as well as Min Zhuang from the laboratory of Jim Wells (UCSF, CA), reported on two distinct approaches to identify substrates for IAP (inhibitor of apoptosis) family of ubiquitin ligases. The Kirkpatrick group used the diGly-antibody enrichment approach in combination with a SMAC-mimetic to specifically inhibit the ubiquitylation of IAP targets in response to apoptosis stimulation. By using cell lines treated with the SMAC-mimetic followed by general caspase inhibition, the Kirkpatrick group identified a group of mitochondrial proteins that are regulated by IAP ligases XIAP and CIAP in response to apoptotic stimuli. Zhuang took a protein engineering approach toward the identification of IAP targets by fusing the NEDD8 specific E2, UBC12 (UBE2M), to the substrate binding domain of either XIAP or CIAP. Incubation of cell lysates with His-Bio-tagged (containing a poly-histidine and peptide sequence for biotinylation) NEDD8 in the presence and absence of these engineered ligases followed by purification of His-Bio-neddylated proteins allowed for the differential identification of proteins that were modified by the engineered ligase. Among the potential candidates identified by this approach, Zhuang identified the mitochondrial localized protein PGAM5 as a new substrate for XIAP. Interesting, only the proteolytically cleaved product of PGAM5 was a substrate for XIAP suggesting a two-step process in targeting PGAM5 for destruction by IAP family ligases. The faithful segregation of the genetic material during cell division via the separation of sister chromatids during mitosis is regulated by APC (anaphase promoting complex)-mediated ubiquitylation of a host of mitotic substrates. To identify new APC substrates, Hanno Steen (Children's Hospital Boston, MA) used a quantitative mass spectrometry approach in which cell lysates from various time points during mitosis were differentially labeled, postdigestion, using tandem-mass tags (TMT). Then the abundance of thousands of proteins were monitored and quantified relative to time points at the onset of mitosis. This approach identified known APC substrates, like securin, as well as novel substrates like the kinesin KIFC1. The advantage of this approach is that it interrogates protein abundance directly and does not require metabolic labeling of cell lines enabling this approach to be applied to a wide range of protein samples including human patient tissue. Proteomics encompasses a variety of approaches toward the systematic study of protein function. Although mass spectrometry based approaches were the dominant theme of the conference, Daniela Rotin (SickKids Hospital, Canada) presented data using protein arrays toward the identification of substrates for HECT E3 ligases. Rotin and colleagues identified FGFR1 as a substrate for the HECT domain ligase Nedd4–1 and characterized the mechanism of Nedd4–1 mediated regulation of FGFR1 signaling. Deletion of the non-canonical Nedd4 binding motif in FGFR1 (i.e. dissimilar to the PY motif) resulted in of FGFR1 signaling and of targets the critical of Nedd4–1 mediated ubiquitylation in FGFR1 and from from the laboratory of (University of detailed preliminary studies using targeted peptide-based arrays to the sequence of various deubiquitylating enzymes. Although identification of substrates for via was a focus of the conference, Ben Cravatt (Scripps Research Institute, CA) reported on their efforts to sites of from Cravatt presented the to identify both the protein substrates that are and the of by using followed by mass spectrometry. 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However, these studies have been to experiments using or tissue culture (University of reported on their studies using metabolic labeling to the in vivo degradation of a large fraction of the proteome within both and mouse These studies allowed for the of the degradation of of proteins. from the studies that in to mass during protein degradation are suggesting that global protein degradation pathways may be noted two important from the of their metabolic labeling studies, including in degradation not the of the amino acid suggesting that the does not global degradation may be a pathway to specific proteins. noted that in degradation are to be the dominant of regulation for most pathways of the all or that have been and large in the degradation of critical signaling proteins have been of degradation for the of proteins are needed to understand the impact of protein degradation pathways. from the of (Scripps Research Institute, CA) reported on the discovery of proteins that were identified using labeling followed by mass spectrometry labeling was by a two a was via with a The from the were for post and to an for or of the number of proteins that in the brain after or revealed that proteins peptides in the brain even after of from the and colleagues found that and the of the complex also the of an low degradation These types of labeling studies the nature of based proteomics on the study of protein degradation pathways. 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