Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

Abstract

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins.

Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome

“…The destabilized Rpn4 substrate had both an I25A and L89G mutation, whereas the yODC substrate had just the L89G mutation (73,74). UbiCRest plasmids expressing deubiquitinases STAM2-AMSH (AMSH*), UBE2D2-OTUB1 (oTUB1*), Cezanne (Addgene plasmid number 61581), and vOTU (Addgene plasmid number 61589) were gifts from David Komander (53,54).…”

“…5b). We therefore turned to UbiCRest analysis, in which a substrate is presented to linkage-specific deubiquitinases (DUBs) to probe its chain composition (53). An Rsp5-or Keap1-ubiquitinated Neh2Dual-Barnase-DHFR substrate was digested by a nonspecific (vOTU), K11-specific (Cezanne), K48-specific (oTUB1* (54)), or K63-specific (AMSH* (54)) DUB.…”

Proteasome‐independent functions of lysine‐63 polyubiquitination in plants

“…The power of clustered regularly interspaced short palindromic repeats (CRISPR) genome editing should, however, help the plant community to develop better tools in the future to tackle the complexity of Ub linkage types in vivo. Chain profiling of a given protein can also be assessed by taking advantage of linkage-specific DUBs (Hospenthal et al, 2015). The availability of DUBs showing diverse specificity and range of activity allows us to probe the entire catalog of modifications.…”

Proteomic techniques to probe the ubiquitin landscape

“…For example, the multiple forms of a protein of several hundred kDa with a conjugated tetraubiquitin can be readily resolved, and one might use a toolbox of specific DUBs [119] to probe the connectivity present in a stepwise manner. This represents a true frontier of ubiquitin proteomics.…”

“…The Ubiquitin Chain Restriction (UbiCRest) assay ( Figure 4F) introduced by the Komander group provides a means of systematically probing a polyubiquitin chain with a toolbox of DUBs of known specificity and using gel-based analysis to determine the linkage varieties present as well as the architecture of a heterotypic polyubiquitin chain [119]. Assumptions are made about the activity of the DUBs, the specificity of which was largely tested using di-ubiquitin, and which may have altered specificities under different concentrations and incubation times or may utilize additional binding domains or interaction partners.…”