SUMMARY Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue’s unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the ‘typical’ phosphoprotein is widely expressed, yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community.

Summary Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform, called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel non-reciprocal proteomic datasets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, sub-cellular localization and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. While conserved protein kinase, lipid kinase, and ubiquitin-like (UBL) protein conjugation sub-networks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here, we report a proteomic analysis of the autophagy interaction network (AIN) in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 4 09 candidate interacting proteins with extensive connectivity among sub-networks. Many new AIN components have roles in vesicle trafficking, protein or lipid phosphorylation, and protein ubiquitination, and affect autophagosome number or flux when depleted by RNAi. The six ATG8 orthologs in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions (LIR) in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

Summary Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly) containing isopeptides following trypsin digestion. We identify ~19,000 diGly modified lysine residues within ~ 5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition indicating both a dependence of on-going translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

SUMMARY Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally-related proteins. Finally, BioPlex, in combination with other approaches can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial Amyotrophic Lateral Sclerosis perturb a defined community of interactors.