Diverse fate of ubiquitin chain moieties: The proximal is degraded with the target, and the distal protects the proximal from removal and recycles

Abstract

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Cellular functions and molecular mechanisms of non-lysine ubiquitination

“…For further consideration, if no DUB can cleave the proximal ubiquitin moiety attached to serine/threonine, ubiquitin would have to be degraded together with the substrate (figure 3 a ). Recent work has demonstrated that the proteasome is, indeed, capable of degrading the proximal ubiquitin along with the substrate [93,94]. Alternatively, the serine/threonine monoubiquitination may serve a signalling function (figure 3 a ).…”

Uncovering the modified immunopeptidome reveals insights into principles of PTM-driven antigenicity

“…The capability to search large number of PTMs allowed us to identify types of modifications that were not examined before in the context of antigen presentation. For example, recent studies have suggested that the proximal ubiquitin may undergo proteasome degradation with its substrate 46,62 . Indeed, we could detect some remnants of ubiquitin-like modifications in our analyses, suggesting that they may be loaded and presented on MHC I and may serve as bone-fide antigens.…”

“…Another interesting group of peptides are those modified with ubiquitin (UB) or ubiquitinlike (UBL) tails (Figure 2A). These are likely a result of the ubiquitin undergoing degradation with its substrate, leaving several residues as a remnant modification 46 . With the aid of PROMISE, we were able to identify eight different remnant mass shifts of different lengths derived from at least three different UB/UBL types across multiple datasets (Supp.…”

Peptide Presentation to T Cells: Solving the Immunogenic Puzzle

“…as a key limiting step in proteasome activity) and turnover of antigens favor a hypothesis of the thymus presenting an altered peptide landscape, ultimately impacting T cell education . All of this work may be further compounded by how proteasomal digests are carried out and assessed, as synthetic peptide substrates may not yield the same products as synthetic proteins, and ideally should factor in substrate ubiquitination …”