The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors

Abstract

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co- and post-translational processing. Using an unbiased biochemical approach we found that UCHL3, USP9X, USP7, USP5 and Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway.

USP5 Is Dispensable for Monoubiquitin Maintenance in Drosophila

“…Therefore, the process of protein ubiquitination and ubiquitin availability both are carefully monitored (1)(2)(3)47). Important regulators of protein ubiquitination are the DUBs, one of which, USP5, has been proposed to function in part by maintaining monoubiquitin (3,7,15,16). Here, our primary question was whether the fly orthologue of this protease is important for monoubiquitin recycling in vivo.…”

“…Monoubiquitin is maintained through de novo synthesis from genes that encode it as a fusion protein to additional ubiquitin moieties or to other proteins and by being cleaved from ubiquitin chains or substrates (15). We wondered whether knockdown of DmUSP5 leads to increased transcription of Primers used rp49 ubiquitin-encoding genes in Drosophila.…”

“…is considered to be primarily a ubiquitin chain dismantler for the purpose of monoubiquitin recycling (3,7,15,16). To examine the effect of DmUSP5 knockdown on overall ubiquitin species in developing flies, we performed Western blotting from whole larval and pupal lysates and probed for ubiquitin (Fig.…”

“…USP5 processes this unanchored chain to yield monoubiquitin (3,10). Thus, this DUB is thought of as a ubiquitin recycler, helping to maintain a monoubiquitin pool for reutilization (7,15,16). Most ubiquitin is found in conjugated forms in various tissues tested, leaving only a small portion available in the unconjugated, monoubiquitin pool (16 -18).…”

A phosphorylation–deubiquitination cascade regulates the BRCA2–RAD51 axis in homologous recombination

“…However, one of the hits, UCHL3, has not been implicated in DNA repair. UCHL3 is a deubiquitination enzyme that is involved in the processing of both Ub precursors (Grou et al 2015) and poly-Ub chain from substrates (Roff et al 1996;Misaghi et al 2005;Kim et al 2011) and has been implicated in several cellular processes, such as oocyte maturation, spermatogenesis, osteoblast differentiation, lipogenesis, retinal degeneration, and epithelial-to-mesenchymal transition (Sano et al 2006;Yi et al 2007;Suzuki et al 2009;Kim et al 2011;Mtango et al 2012a,b;van Beekum et al 2012;Song et al 2014). However, the mechanism of UCHL3's functions remains unclear, as only a limited number of UCHL3 targets were identified.…”