S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Abstract

Ubiquitin (Ub)-protein conjugates formed by purified ringfinger or U-box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST-fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST-UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non-forked Ub conjugates.

A compact sunflower line produced after cross Helianthus annuus x Verbesina encelioides

“…Several comprehensive works reported a good correlation between phenolic compounds and total antioxidants [25,26]. The total tocopherol content was reduced 2-fold in the hybrid plants compared with the cultivated sunflower ( Table 2).…”

Disassembly of Lys11 and Mixed Linkage Polyubiquitin Conjugates Provides Insights into Function of Proteasomal Deubiquitinases Rpn11 and Ubp6

“…Given that we have now uncovered an ability of proteasomes to rapidly disassemble Lys 11 linkages, it is unclear how they may serve as a targeting signal (17,18,75,76 (18). Nevertheless, linkage branching should be considered on a case-by-case basis, given that some types of forked linkages have been reported to inhibit proteasomal degradation (79), whereas a Lys 48 /Lys 63 -linked branched chain had no effect on deconjugation (48).…”

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

“…This in vitro finding is consistent with previous observations that several physiologically important E3s, including a U-box E3 (CHIP) and RING finger E3s (MuRF1 and MDM2), form polyubiquitin chains that contain all seven types of isopeptide linkage and which are also forked (Lys 6 plus Lys 11 , Lys 27 plus Lys 29 , and Lys 29 plus Lys 33 ) (62). One possible explanation for the different fates of polyubiquitylated proteins is that cells contain additional auxiliary factors, such as S5a (also known as RPN10), that block the formation of nondegradable polyubiquitin chains to facilitate substrate degradation (62)(63)(64). We speculate that DDX24 might escape from these factors by physical or spatial sequestration, resulting in a fate different from that of other substrates, such as p53.…”