Defects in ubiquitin (Ub) pathways are often responsible for cancer and devastating neurodegenerative diseases. Because of its central role in biological circuits and the potential for therapeutic intervention, the Ub system is an intense research area. However, Ub biology is highly complex, e.g. in humans it is supported by over 500 Ub ligases and 95 deubiquitinases (DUBs). Currently, this complexity and our limited understanding of players and their interactions are a severe hindrance for targeted intervention for many diseases. Recent advances in mass spectrometry (MS)-based technologies open up the exciting possibility to characterize the entire Ub information network in an unbiased and global manner. While in the long term our studies will be performed in human cells, we will first characterize this network in yeast, which allows comparatively easy global proteomic measurements and protein expression manipulations. We will collect global measurements surrounding each of ~120 genes implicated as Ub-dependent enzymes (all E3 ligases, F-box proteins, DUBs). We term this the UR120 set for """"""""ubiquitin-regulatory"""""""" genes. The collection of hundreds of global datasets requires multiplexing to sustain throughput. Specifically, in Aim 1, we will profile Ub conjugates across individual UR120 deletion strains including a panel of ten conditions where ubiquitylation plays specific roles in cellular response and regulation, thus identifying ubiquitylation events occurring in a condition-dependent manner.
In Aim 2, deletions strains for the UR120 genes will be globally profiled for protein expression under the same ten conditions as in Aim 1 to detect stable and dynamic changes in protein expression.
In Aim 3, we will develop a pipeline for the large-scale analysis of protein interactions specific to the Ub pathway. A library of the UR120 genes will be created with HA-tagged proteins under endogenous expression levels and their interactions identified by affinity purification (AP)-MS. The same ten conditions will be examined to learn how the UR120 interaction network responds to changes in cellular environment. We will also perform AP-MS analyses for inactive variants of ligases and dequbiquitinases to stabilize enzyme-substrate pairing. The final result of this work will be a three-pronged information network comprised of conjugate profiling, expression profiling, and physical interaction studies. From this, we will prepare a first-of-its-kind draft o a comprehensive map of all potential enzyme-substrate relationships for ligases and DUBs. By examining the UR120 set under multiple conditions directly relevant to Ub biology, we can begin to understand how different elements cooperate throughout a normal cell-cycle and in response to perturbations. These data will provide a comprehensive assessment of the protein ubiquitylation profiles and can be used to uncover enzyme-substrate relationships. Upon completion, our next step will be to create the complementary information networks for human genes.

Public Health Relevance

The ubiquitin system is an intense research area with great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in ubiquitin biology have been implicated as a contributing factor in the causation of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. This proposal will apply mass spectrometry-based technologies to systematically map the substrates of every enzyme in the yeast ubiquitin system (all ligases and deubiquitinases).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Membrane Biology and Protein Processing (MBPP)
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Gerratana, Barbara
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Harvard University
Anatomy/Cell Biology
Schools of Medicine
United States
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Erickson, Brian K; Rose, Christopher M; Braun, Craig R et al. (2017) A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization. Mol Cell 65:361-370
QuirĂ³s, Pedro M; Prado, Miguel A; Zamboni, Nicola et al. (2017) Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals. J Cell Biol 216:2027-2045
Bai, Lin; Jastrab, Jordan B; Isasa, Marta et al. (2017) Structural Analysis of Mycobacterium tuberculosis Homologues of the Eukaryotic Proteasome Assembly Chaperone 2 (PAC2). J Bacteriol 199:
Lee, Ho-Joon; Jedrychowski, Mark P; Vinayagam, Arunachalam et al. (2017) Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation. Cell Rep 20:721-736
Whiteley, Alexandra M; Prado, Miguel A; Peng, Ivan et al. (2017) Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins. Elife 6:
Everley, Robert A; Huttlin, Edward L; Erickson, Alison R et al. (2017) Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags. J Proteome Res 16:1069-1076
Ersing, Ina; Nobre, Luis; Wang, Liang Wei et al. (2017) A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells. Cell Rep 19:1479-1493
Rose, Christopher M; Isasa, Marta; Ordureau, Alban et al. (2016) Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes. Cell Syst 3:395-403.e4
Chick, Joel M; Munger, Steven C; Simecek, Petr et al. (2016) Defining the consequences of genetic variation on a proteome-wide scale. Nature 534:500-5
Paulo, Joao A; O'Connell, Jeremy D; Everley, Robert A et al. (2016) Quantitative mass spectrometry-based multiplexing compares the abundance of 5000 S. cerevisiae proteins across 10 carbon sources. J Proteomics 148:85-93

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