To determine whether IU1 is cell permeable, it was added to cultures and cell-associated IU1 was quantified by LC-MS or UV absorption. When added at 50 μM, IU1 reached an apparent intracellular concentration of ~13 μM within 1 hour, which remained constant over the course of the experiment (Supplementary Figs. 17–19
). Effects of IU1 on the viability of MEFs only became apparent at 250μM (Supplementary Figs. 20, 21
). Moreover, IU1 did not noticeably induce apoptosis (Supplementary Fig. 22
). When cell proliferation was measured in real time, slight inhibition became apparent at 120 μM (Supplementary Fig. 21
). In the case of both cell viability and proliferation assays, usp14−/−
MEFs were no less sensitive than wild-type, indicating that IU1 toxicity at high concentrations was independent of Usp14 inhibition.
To determine whether IU1 could enhance proteasome function in cells, we expressed Tau in MEFs treated with sub-cytotoxic doses of IU1. IU1 induced dose-dependent reduction in Tau levels, with a strong effect seen at 50 μM (; Supplementary Fig. 23
). Thus, IU1 treatment affected Tau similarly to Usp14-CA (), consistent with active site inhibition. No effect was seen on Tau mRNA (Supplementary Fig. 24
). When usp14−/−
MEFs were treated with IU1, no effect on Tau was observed, indicating that IU1 enhances Tau degradation through inhibiting Usp14 (). Based on these and previous experiments (), nonspecific inhibition of other DUBs by IU1 does not affect proteasome function at this dose. The effect of IU1 on Tau degradation was independent of autophagy (Supplementary Fig. 25
). Several other proteins implicated in proteotoxic mechanisms–TDP-43, ataxin-3, and glial fibrillary acidic protein (GFAP)–were similarly depleted from MEFs by IU1 (; Supplementary Fig. 26
). The effectiveness of IU1 in neurons, where proteotoxic mechanisms are commonly observed, has not been examined.
IU1 enhances proteasomal degradation in vivo
IU1 enhanced the extent of ubiquitin modification of TDP-43 in cells, perhaps accounting for its accelerated degradation (). In contrast, little change was seen in bulk cellular ubiquitin conjugates (; Supplementary Fig. 27
). Free ubiquitin was reduced following IU1 addition, and, as the dose of IU1 increased, the level of free ubiquitin in wild-type MEFs approached that of untreated usp14−/−
MEFs (). Previous work showed that Usp14 and Ubp6 assist in maintaining of cellular ubiquitin pools by suppressing proteasomal degradation of ubiquitin11,13,14,17,34,35
. The conjugated rather than free form of ubiquitin is most subject to degradation36
. By separating ubiquitin from its conjugative target, Usp14 antagonizes this pathway of ubiquitin degradation.
Enhanced protein degradation in cells treated with IU1 could result from increased synthesis of proteasomes; however, no significant changes in proteasome composition were seen after IU1 treatment (Supplementary Fig. 28
). Usp14 is known to affect gating of the proteasome21
, but this does not appear to be critical in the mode of action of IU1 (unpublished data). The detailed similarities observed between the effects of mutational inactivation of Usp14’s catalytic site and IU1 treatment, as well as the observation that Usp14 is required for IU1 to affect protein degradation, provide strong evidence for the importance of chain trimming by Usp14. In addition, IU1 had little or no effect on the in vivo degradation of a ubiquitin-independent substrate37
of the proteasome, cODC-EGFP (). Similar results were obtained in vitro with antizyme-promoted ODC degradation (data not shown). The effects of IU1 are likely restricted to ubiquitin-dependent proteasome substrates, based on its mode of action, but further characterization is required to establish this. Finally, Arg-GFP levels were constant upon treatment with IU1, when assayed in cells expressing Usp14-CA, suggesting that IU1 does not influence Usp14’s noncatalytic inhibitory effect (Supplementary Fig. 26
Oxidized proteins form a class of misfolded proteasome substrates that increase with age and are apparently toxic when they accumulate38,39
. We induced protein oxidation by treating cells with menadione, and visualized oxidized species via their carbonyl groups. IU1 treatment strongly reduced the accumulation of oxidized proteins (). When proteasome inhibitor was added, the effect of IU1 was attenuated, suggesting that IU1 does not prevent the oxidation reaction itself. IU1 treatment reduced menadione toxicity substantially in HEK293 cells (), strongly supporting the hypothesis that proteins are critical targets of oxidative damage. IU1 also reduced the toxicity of an unrelated oxidizing agent, hydrogen peroxide (data not shown). IU1C, the IU1 variant that is inactive against Usp14, failed to reduce menadione cytotoxicity (Supplementary Fig. 29
). In summary, these experiments suggest that IU1 can promote cell survival during proteotoxic stress.
IU1 alleviates cytotoxicity induced by oxidative stress