Assembly and disassembly of branched ubiquitin chains

Abstract

Protein ubiquitylation is an essential post-translational modification that regulates nearly all aspects of eukaryotic cell biology. A diverse collection of ubiquitylation signals, including an extensive repertoire of polymeric ubiquitin chains, leads to a range of different functional outcomes for the target protein. Recent studies have shown that ubiquitin chains can be branched and that branched chains have a direct impact on the stability or the activity of the target proteins they are attached to. In this mini review, we discuss the mechanisms that control the assembly and disassembly of branched chains by the enzymes of the ubiquitylation and deubiquitylation machinery. Existing knowledge regarding the activities of chain branching ubiquitin ligases and the deubiquitylases responsible for cleaving branched chains is summarized. We also highlight new findings concerning the formation of branched chains in response to small molecules that induce the degradation of otherwise stable proteins and examine the selective debranching of heterotypic chains by the proteasome-bound deubiquitylase UCH37.

Keywords: branched ubiquitin chain; proteasome; protein degradation; ubiquitin; ubiquitylation (ubiquitination).

FIGURE 1

Ubiquitin chain architectures and mechanisms of branched chain assembly and disassembly. (A) Ubiquitin chains can be classified as either homotypic or heterotypic (mixed and branched) based on the types of ubiquitin linkages and how the subunits are connected to each other within the chain. Examples of homotypic K48- and K63-linked chains are shown. Other sites of chain formation include M1, K6, K11, T12, S20, T22, K27, K29, K33, and T55. Mixed K11/K48 and M1/K63 chains as well as branched K29/K48 and K48/K63 chains are shown as examples of heterotypic chains. The modified acceptor sites of the ubiquitin subunits are indicated in the figure. The terminal (unmodified) subunits are colored in light grey. (B) Mechanism of branched chain formation by the HECT ubiquitin ligases ITCH and UBR5. ITCH attaches homotypic K63-linked chains to TXNIP. UBR5 then binds to the K63 linkages through its UBA domain to nucleate the formation of K48 linkages, resulting in the assembly of branched K48/K63 chains. (C) PROTAC-dependent formation of branched chains by CRL2^VHL and TRIP12. CRL2^VHL assembles homotypic K48-linked chains on BRD4 and then recruits TRIP12, which attaches K29 linkages and further stimulates CRL2^VHL activity. This cascade leads to the formation of complex branched K29/K48 chain architectures on BRD4. CLR2^VHL subunits include VHL, elongin B (ELOB), elongin C (ELOC), CUL2, RBX1, and E2. (D) Mechanism of branched chain disassembly by the proteasome-bound UCH37/RPN13 complex. UCH37/RPN13 binds to both distal ubiquitin subunits that emanate from the K6/K48 branch point. Cleavage of the K48 linkage at the branch point is greatly stimulated by RPN13, resulting in trimming of the chain at the proximal K48 linkage and the release of homotypic K6- and K48-linked chains. The K6-linked chain attached to MFN2 is then presumably cleaved en bloc by RPN11, an essential proteasome-associated DUB component of the 19S regulatory particle, prior to MFN2 degradation. MFN2 was identified as a substrate of UCH37 in Du et al. (2022).