Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome

Abstract

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.

The role of K63‐linked polyubiquitination in cardiac hypertrophy

“…The HECT E3 ligases transiently bind with Ub by forming a thioester intermediate, before Ub transferred to substrate, but RING ligases act as scaffold, and it promotes the direct transfer of Ub to substrate by bringing the E2‐bound Ub close to substrate 13. The proteomic studies have shown that all types of polyubiquitination co‐exist in cells 14, 15. Lys48‐linked chains are the most abundant linkage type (often > 50% of all linkages) found in cells that are primarily involved in transfer of substrate proteins to the 26S proteasome for degradation 16.…”

Proteomic techniques to probe the ubiquitin landscape

“…This technique was used in three landmark papers from the Gygi, Mann and Elledge laboratories to identify the largest set, to date, of ubiquitin substrates [41,89,90]. Although the identification rate was initially in the range of 300 to 800 ubiquitination sites [81,88] employment of peptide fractionation, such as isoelectric focusing or strong cation exchange chromatography, improved the identification rates to 10000 -20000 ubiquitination sites in one study [41,[91][92][93].…”

“…Although the identification rate was initially in the range of 300 to 800 ubiquitination sites [81,88] employment of peptide fractionation, such as isoelectric focusing or strong cation exchange chromatography, improved the identification rates to 10000 -20000 ubiquitination sites in one study [41,[91][92][93]. Using β-interferon stimulation to stimulate ISG15 expression as a positive control, Kim et al estimated the contribution of ISGylation (Figure 2) of substrates to the total number of di-glycine modified peptides, and concluded that a negligible portion of the total modified peptide population can be attributed to ISG15 modification [89]. In the same study, a general DUB was used to cleave all ubiquitin moieties from their substrates leaving only the sites modified by Nedd8.…”

“…In the same study, a general DUB was used to cleave all ubiquitin moieties from their substrates leaving only the sites modified by Nedd8. Once again the group concluded that the fraction of proteins modified by Nedd8 was minor [89].…”

Protein monoubiquitylation: targets and diverse functions

“…1997; Kim et al . 2011). In this review, we present recent findings related to monoubiquitylation, with a focus on the best‐studied mammalian systems, and we provide an overview of monoubiquitylated proteins and the effects of monoubiquitylation on substrates categorized according to their function.…”