Extended Data Fig. 3: Characterization of the deubiquitylase USP20.
From: Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis
a, In vitro activities of USP20 variants indicated by the hydrolysis of a fluorogenic substrate Ubiquitin-7-amino-4-trifluoromethylcoumarin (Ub-AFC). The WT USP20 and USP20(C154S) proteins were purified from HEK 293T cells. Negative control (NC) means no protein added. Inset western blotting shows the similar amount of the proteins. b, Hydrolysis of various linked di-ubiquitin (Ub2) by USP20 in vitro. c, USP20 decreases sterol-induced ubiquitination of HMGCR. CHO-7 cells were transfected with the indicated plasmids, depleted of sterols as in Extended Data Fig. 2 and then treated with 25-HC plus 10 μM MG132 for 2 h. Cells lysates were immunoprecipitated with anti-T7 beads. The pellet was immunoblotted with anti-HA antibody and anti-HMGCR. The input was immunoblotted with anti-FLAG. d, GSK2643943A inhibits USP20-mediated deubiquitination of HMGCR. CHO-7 cells were transfected with plasmids and depleted of sterols. Then the cells were treated with 1 μg ml-1 25-HC in the presence of MG132 for 2 h. Cell lysates were immunoprecipitated with anti-T7 beads. The pellet was immunoblotted with anti-HA and anti-HMGCR antibodies. The input was immunoblotted with anti-FLAG. e, GSK2643943A inhibits USP20-mediated HMGCR deubiquitination in vitro. The sterol-depleted CHO-7 cells were treated with 25-HC plus MG132. Cells lysates were immunoprecipitated with anti-HMGCR antibody-coupled agarose. The pellet samples were then incubated with recombinant USP20 protein for 30 min at 37 °C. *: non-specific band. USP20 was inactivated by boiling for 10 min as a control. Experiments in (a–e) were performed as indicated three times with similar results. f–h, Knockdown of Usp20 accelerated sterol-induced degradation of HMGCR. Huh7 cells were transfected with scrambled (negative control, NC) or USP20-targeting siRNA, treated as in Fig. 1d. The experiments were repeated for three times. i, Quantification of HMGCR in (f–h). Data are mean ± SEM and were analysed by two-way ANOVA.
