Enhancement of proteasome activity by a small-molecule inhibitor of USP14

Abstract

Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that Usp14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates, in vivo and in vitro. A catalytically inactive variant of Usp14 has reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human Usp14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. Usp14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of Usp14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?

“…A key experiment from Lee et al , 2010, (Figure 1g) showed that recombinantly expressed tau or TDP-43 levels in HEK293 cells were higher when coexpressed with wild type as compared to catalytically inactive (C114A) USP14. We cotransfected V5-pTT5d-USP14 or V5-pTT5d-USP14 (C114A) plasmids (ranging from 0.5 to 2µg) and 2µg pTT5d-Tau or pTT5d-Flag-TDP-43 plasmids in HEK293 cells.…”

“…Lee et al (2010) showed that Usp14 -/- mouse embryonic fibroblasts had lower levels of tau or TDP-43 than those overexpressing wild-type USP14. Therefore, we reasoned that USP14 knockdown should result in lower levels of substrate.…”

“…This mutual interaction of USP14/Ubp6 with the proteasome is thought to enhance selectivity of the proteasome for ubiquitinated proteins and couple deubiquitination to degradation. In contrast, Finley and colleagues found that USP14/Ubp6, and in some instances a catalytically inactive mutant (C114A in mammals), could cause an inhibition of the degradation of substrates ( Hanna et al , 2006; Lee et al , 2010). For model substrates and ataxin3, this effect was shown to require USP14/Ubp6 protein but not its catalytic activity.…”

“…This led to the hypothesis that deubiquitination of substrates by USP14 at a faster rate than the proteasome initiates degradation could cause rejection of otherwise competent substrates from the proteasome. Supporting this hypothesis, inhibition of USP14 by a small molecule inhibitor (IU1) enhanced proteasomal substrate degradation in cells overexpressing tau or TDP-43 ( Lee et al , 2010). Thus, inhibition of USP14 was proposed as a therapeutic strategy to enhance proteasomal function in neurodegenerative diseases in which these proteins accumulate.…”

“…For model substrates and ataxin3, this effect was shown to require USP14/Ubp6 protein but not its catalytic activity. For two proteins involved in neurodegenerative diseases, tau and TDP-43, inhibition of proteasomal degradation by USP14 was dependent on its deubiquitinating activity, since the catalytically inactive mutant had no effect ( Lee et al , 2010). This led to the hypothesis that deubiquitination of substrates by USP14 at a faster rate than the proteasome initiates degradation could cause rejection of otherwise competent substrates from the proteasome.…”