Synthetic Uncleavable Ubiquitinated Proteins Dissect Proteasome Deubiquitination and Degradation, and Highlight Distinctive Fate of Tetraubiquitin

Abstract

Various hypotheses have been proposed regarding how chain length, linkage type, position on substrate, and susceptibility to deubiquitinases (DUBs) affect processing of different substrates by proteasome. Here we report a new strategy for the chemical synthesis of ubiquitinated proteins to generate a set of well-defined conjugates bearing an oxime bond between the chain and the substrate. We confirmed that this isopeptide replacement is resistant to DUBs and to shaving by proteasome. Analyzing products generated by proteasomes ranked how chain length governed degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetra-ubiquitin chain can be degraded with the substrate. Together these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.

The Logic of the 26S Proteasome

“…These findings imply that the presence of a single Ub or short chains on the substrate do not prevent its translocation and destruction (Singh et al, 2016). However, removing large Ub chains from the substrate must be important for substrate translocation because a long Ub chain should prevent efficient passage through the ATPase ring into the 20S.…”

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

“…In parallel, proteasome-associated deubiquitinases (DUBs) remove the polyUb signal from the substrate (Finley, 2009; Glickman and Adir, 2004; Guterman and Glickman, 2004; Lee et al, 2011; Mansour et al, 2015). DUB and ATPase activities are carefully coordinated in the 19S RP to allow for proteolytic efficiency and recycling of ubiquitin (Aufderheide et al, 2015; Matyskiela et al, 2013; Peth et al, 2009; Peth et al, 2013a, b; Peth et al, 2013d; Singh et al, 2016; Verma et al, 2002; Verma et al, 2000). These activities are coordinated, to a large extent, by the two largest subunits in the 19S RP, Rpn1 and Rpn2 that function as flexible scaffolds.…”

Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags

“…17–19 This has facilitated biochemical, biophysical and functional analyses of these conjugates. 18–20 Equipped with various synthetic tools for protein synthesis of such complex conjugates, we decided to examine the total chemical synthesis of SUMO-2 linked to Lys63-di-Ub via its N-terminus, di-Ub(K63)-SUMO-2 ( 1 , Scheme 1), using a convergent approach.…”