The ubiquitin interacting motifs of USP37 act on the proximal Ub of a di-Ub chain to enhance catalytic efficiency

Abstract

USP37 is a deubiquitinase (DUB) with roles in the regulation of DNA damage repair and the cohesion of sister chromatids during mitosis. USP37 contains a unique insert of three ubiquitin interacting motifs (UIMs) within its catalytic DUB domain. We investigated the role of the three UIMs in the ability of USP37 to cleave di-ubiquitin chains. We found that the third UIM of USP37 recognizes the proximal ubiquitin moiety of K48 di-Ub to potentiate cleavage activity and posit that this mechanism of action may be generalizable to other chain types. In the case of K48-linked ubiquitin chains this potentiation stemmed largely from a dramatic increase in catalytic rate (k_cat). We also developed and characterized three ubiquitin variant (UbV) inhibitors that selectively engage distinct binding sites in USP37. In addition to validating the deduced functional roles of the three UIMs in catalysis, the UbVs highlight a novel and effective means to selectively inhibit members of the difficult to drug DUB family.

Figure 1

Di-Ub cleavage specificity of USP37^wt and USP37^mUIM1,2,3. (a) Domain architecture of human and zebrafish USP37. (b) Deubiqutination time course for USP37^wt (top panel) and USP37^mUIM1,2,3 (bottom panel) towards each of the 8 di-Ub linkages. Assay was performed in three independent experiments and representative gels are shown. Black lines delineate the separation of distinct gels. USP37^wt and USP37^mUIM1,2,3 were used at a concentration of 1 nM. Uncropped versions of gels are shown in Supplementary Fig. S1.

Figure 2

Activity of USP37^wt and USP37 mutants towards IQF K11, K48, and K63 probes. (a) Michaelis-Menten kinetic analysis of USP37^wt and USP37^mUIM1,2,3 for the substrates K11-, K48- and K63-linked IQF di-Ubs. Curves represent measurements from three independent experiments each measured in duplicate. Values reported are mean ± SD. (b) Progress curves for USP37^wt and USP37 mutants towards IQF K11-, K48- and K63-linked di-Ubs. Assay was performed in duplicate in three independent experiments and representative curves are shown. For both experiments, USP37 was used at 25, 1.25 or 12.5 nM for K11, K48 or K63 di-Ub chains, respectively.

Figure 3

USP37^wt and USP37^mUIM1,2,3 activity towards Ub-AMC. (a) Cartoon models of di-Ub and Ub-AMC illustrating how di-Ub contains both a distal and proximal Ub, while Ub-AMC contains only a distal Ub. (b) Cartoon model of the two possibilities of how the UIMs of USP37 can interact with Ub-AMC and the expected effects of each on Ub-AMC hydrolysis. The cartoon depicts a model in which the UIMs bind to the same di-Ub chain displayed across the enzyme active site. (c) Progress curve for USP37^wt and USP37^mUIM1,2,3 activity against Ub-AMC. Assays were performed in duplicate in three independent experiments and representative curves are shown. Enzymes were used at a concentration of 0.5 nM.

Figure 4

USP37^wt and USP37^mUIM1,2,3 activity towards proximally mutated K48 di-Ub. (a) Cartoon model for how USP37^wt and USP37^mUIM1,2,3 are expected to cleave K48 and proximally mutated K48 chains. The cartoon depicts a model in which the UIMs bind to the same di-Ub chain displayed across the enzyme active site. (b) Deubiquitination time course for USP37^wt (top panel) and USP37^mUIM1,2,3 (bottom panels) towards K48, K48^I44A, and K48^I44W di-Ub. Assay was performed in three independent experiments and representative gels are shown. USP37 enzymes were used at a concentration of 5 nM. Black boxes delineate distinct gels or cropped areas of same gel. Uncropped versions of gels are shown in Supplementary Fig. S1.

Figure 5

Analysis of inhibitory effects of UbVs on USP37 activity. (a) Inhibition of USP37^wt cleavage of Ub-AMC by UbV.core, UbV.UIM1, and UbV.UIM*. Curves represents measurements from three independent experiments. Values reported are mean ± SD. (b) Inhibition of USP37^wt cleavage of IQF K48-linked di-Ub by UbV.core, UbV.UIM1 and UbV.UIM*. Curves represents measurements from three independent experiments. Values reported are mean ± SD. For both experiments USP37 was used at a concentration of 0.5 nM.