Lys6-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation

Abstract

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys 6 of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys 6 was the most readily labeled with sulfosuccinimidobiotin. Lys 6 -biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys 6 -biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys 6 -biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys 6 had similar phenotypes as Lys 6 -biotinylated ubiquitin. Lys 6 mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys 6 -modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys 6 -modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract

“…It appears that an inability to clear Cx43 conjugates that incorporate K6W-Ub results in their accumulation, a backup of this UPS, and, thus, stabilization of the parent protein as well. This mechanism additionally suggests that expression of K6W-Ub may also compromise and saturate the other UPSs, and it provides further rationale as to why cells in which K6W-Ub is expressed have limited capacity to withstand various stresses, particularly proteotoxicity (8,13). Please see SI Text for discussion of relations between monoubiquitination and calpain function and for possible other proteolytic or nonproteolytic mechanisms regulating Cx43, Cx46.…”

“…Other work showed that Ub mutations in which K6 is replaced by various amino acids, i.e., K6W-Ub, are conjugation competent but proteolytically incompetent. Thus, cells and tissues in which K6W-Ub is expressed accumulate Ub conjugates (13). Exchanging K for A or R has the same effects.…”

“…5B, compare lane 6 to lane 5). Such conjugates would be expected to accumulate because K6W-Ub conjugates resist degradation (13).…”

Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells

“…Timely removal of abnormal or damaged proteins by the UPP is essential for the cells to withstand and recover from various environmental stresses (Dudek et al, 2005;Shang et al, 2005). However, the UPP itself is also a target of such stresses.…”

“…In contrast with many other types of cells, RPE have limited levels of free ubiquitin. Thus, addition of exogenous ubiquitin to RPE supernatant generated significantly higher levels of ubiquitin conjugates and also enhanced the proteasome-dependent proteolysis (Obin et al, 1994(Obin et al, , 1995Shang et al, 2005). The limited levels of endogenous ubiquitin may render the UPP in RPE more vulnerable to a variety of environmental stresses.…”