Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues

Abstract

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.

Human Liver Cytochrome P450 3A4 Ubiquitination

“…It remains to be determined at present whether these are common structural features of the ubiquitination of not only all P450 proteins but also that of an expanding list of other canonical substrates of these particular E2-E3 ubiquitination complexes. The identification FEBRUARY 6, 2015 • VOLUME 290 • NUMBER 6 through Ub remnant profiling approach of Asp/Glu/Ser/Thr as predominant residues flanking ubiquitinated Lys sites in the global protein ubiquitinome considerably strengthens this possibility (81,82). Nevertheless, to our knowledge, our findings provide the very first mechanistic example of UBC7-gp78 substrate recognition.…”

“…In this approach, a monoclonal antibody rather selectively recognizes the Ub-derived diGly remnant (GGK) on the ubiquitinated Lys residues derived from the tryptic digestion of the Ub-modified proteins, enabling the 1000-fold enrichment of GGK-modified peptides, with a characteristic added mass of 114.0429 Da (84). This GGK remnant mass signature of ubiquitinated proteins has enabled the unprecedented global identification of ubiquitinated cellular proteins by LC-MS/MS analyses (80 -84), leading to the identification of linear peptide sequences in this vast "ubiquitinome" in which the ubiquitinated Lys residues are often flanked by Asp/Glu/Ser/Thr residues (81,82).…”

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

“…In fact, all sites that we studied here and the additional sites we found in our in vitro/in vivo KAT assays are targeted by ubiquitylation (41,42). Moreover, succinylation of Ran has been detected in HeLa cells (lysines 23, 37, 99, 127, and 152) and mouse embryonic fibroblasts (lysines 23, 134, 142, and 159) (43,44).…”

Role of the alternative splice variant of NCC in blood pressure control

“…Other posttranslational modifications that play a crucial role in the regulation of NCC activity include ubiquitylation and phosphorylation (reviewed in [27–29]. According to recent large-scale proteomics studies, NCC is ubiquitylated on several conserved lysine residues [30,31]. Generally, NCC ubiquitylation is associated with increased endocytosis from the plasma membrane, as well as protein degradation [32,33].…”