Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo.

Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods. Specifically, we employ tandem ubiquitin binding entities (TUBEs) and the K29-selective Npl4 Zinc Finger 1 (NZF1) domain from the deubiquitinase TRABID to enrich for chains from human cells. Minimal trypsinolysis followed by high resolution mass spectrometric analysis reveals that Ub chain branching can indeed be detected using both Ub binding domains (UBDs) tested at endogenous levels. We find that ∼1% of chains isolated with TUBEs contain Ub branch points, with this value rising to ∼4% after proteasome inhibition. Electron-transfer dissociation (ETD) analysis indicates the presence of K48 in these branched chains. The use of the NZF1 domain reveals that ∼4% of the isolated chains contain branch points with no apparent dependence on proteasome inhibition. Our results demonstrate an effective strategy for detecting and characterizing the dynamics of branched conjugates under different cellular conditions.

Subunit-Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases.

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains.