Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

Abstract

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.

USP1 (ubiquitin specific peptidase 1) targets ULK1 and regulates its cellular compartmentalization and autophagy

“…To characterize the ULK1 puncta observed in USP1depleted cells, we verified their colocalization/interaction with the ubiquitin-binding receptor protein SQSTM1 both by immunofluorescence analysis and immunoprecipitation experiments. As previously shown for WIP1130-dependent GFP-ULK1 aggresomes [20], endogenous ULK1 partly colocalized with SQSTM1 dots (Figure 5(a,c)). Notably, SQSTM1 dots were more abundant in USP1-depleted cells ( Figure 5(a, b)).…”

“…It has been shown that deubiquitinase inhibition by WP1130 reduce the protein level of ULK1 in the Triton X-100 soluble fraction of mammalian cell extracts [20]. In confirmation of this study, USP1 depletion using siRNA, was coupled to a reduction of total ULK1 and ULK1 p-Ser555 in the soluble fraction of U2OS, HEK293, and MCF10AT cells ( Figure 1(df)).…”

“…We confirmed that ULK1 deubiquitnation sharply affects its compartmentalization [20], and showed that USP1 is the responsible DUB. We also demonstrated that USP1 depletion increases the TRAF6-dependent K63-linked ubiquitination of ULK1 triggered by rapamycin addition [18], arguing for a direct involvement of USP1 in the removal of K63-linked ubiquitin from ULK1.…”

“…The DUB inhibitor WP1130 induced relocalization of GFP-ULK1 to aggresome-like structures, as detected by fluorescence analysis, and to a Triton X-100-insoluble pellet upon cells lysis [20]. We verified whether USP1 depletion could have the same effect on endogenous ULK1.…”

“…Conversely, ULK1 ubiquitination by CUL3 (cullin 3) leads to its proteasomedependent degradation and autophagy termination [19]. Notably, deubiquitination inhibition by a chemical compound, WP1130, leads to ULK1 aggregation and blockade of autophagy [20].…”