Resolving the Complexity of Ubiquitin Networks

Abstract

Ubiquitination regulates nearly all cellular processes by coordinated activity of ubiquitin writers (E1, E2, and E3 enzymes), erasers (deubiquitinating enzymes) and readers (proteins that recognize ubiquitinated proteins by their ubiquitin-binding domains). By differentially modifying cellular proteome and by recognizing these ubiquitin modifications, ubiquitination machinery tightly regulates execution of specific cellular events in space and time. Dynamic and complex ubiquitin architecture, ranging from monoubiquitination, multiple monoubiquitination, eight different modes of homotypic and numerous types of heterogeneous polyubiquitin linkages, enables highly dynamic and complex regulation of cellular processes. We discuss available tools and approaches to study ubiquitin networks, including methods for the identification and quantification of ubiquitin-modified substrates, as well as approaches to quantify the length, abundance, linkage type and architecture of different ubiquitin chains. Furthermore, we also summarize the available approaches for the discovery of novel ubiquitin readers and ubiquitin-binding domains, as well as approaches to monitor and visualize activity of ubiquitin conjugation and deconjugation machineries. We also discuss benefits, drawbacks and limitations of available techniques, as well as what is still needed for detailed spatiotemporal dissection of cellular ubiquitination networks.

Ubiquitomics: An Overview and Future

“…In addition to biochemical and antibody-based methods [ 8 ], mass spectrometry (MS)-based proteomics has enabled the rise of “ubiquitomics”, a term first coined in 2007 [ 9 ]. A ubiquitome refers to the set of proteins that are modified by ubiquitin and the associated ubiquitin chain topologies found under these conditions, although it is not possible to determine both of these aspects in the same experiment.…”

The Ubiquitin System and A20: Implications in Health and Disease

“…Ubiquitination is a highly dynamic, enzymatically catalyzed posttranslational modification, which regulates cellular and immune homeostasis through structurally and functionally distinct polyubiquitin signals (Swatek and Komander 2016; Kliza and Husnjak 2020; Mendes et al 2020). Ubiquitin can be covalently attached to targeted substrates as monomers (monoubiquitination) or polymers (polyubiquitination) through the coordinated activity of 3 main enzymes: E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligating).…”

“…Among many regulatory circuits at work, ubiquitination has recently emerged as an essential player in controlling virtually every aspect of cellular homeostasis (Swatek and Komander 2016; Rape 2018; Kliza and Husnjak 2020). In addition, aberrant function of enzymes involved in ubiquitin regulation oftentimes contribute to the onset and progression of several disorders.…”

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer

“…We also identified TCHP as a substrate of USP8 in the regulation of ciliogenesis [ 34 ], but the substrates for the other five DUBs are unknown. A variety of experimental approaches have been developed to identify DUB substrates [ 165 ]. Stable overexpression or knockdown of DUBs followed by quantitative proteomic analysis to detect proteins differentially expressed in control and manipulated cells identified Sec28p and NFX1-123 as substrates of Ubp3p and USP9X, respectively [ 166 , 167 ].…”

“…The screening of compounds for effects on protein ubiquitination and deubiquitination has led to the identification of a number of E3 ubiquitin ligase- and DUB-targeting drugs [ 29 , 165 , 177 ]. Because the substrates of these enzymes have a wide variety of functions, such drugs can interfere with various signaling pathways and impair physiological functions.…”