OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

Abstract

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1^-/- knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

Keywords: UBE2E1; deubiquitylation (deubiquitination); histone; ubiquitin; ubiquitin thioesterase (OTUB1); ubiquitin-conjugating enzyme (E2 enzyme).

Figure 1.

UBE2E1 levels are reduced in OTUB1 knockout MEFs. A, tandem mass tag MS analysis of MEF WT and OTUB1^−/− knockout cells. B, Western blotting of the whole cell lysate of MEF WT and OTUB1^−/− knockout cells with the indicated antibodies.

Figure 2.

OTUB1 regulates the stability of UBE2E1 protein levels. A, Western blot analysis of the whole cell lysate of U2OS wildtype (WT) and CRISPR-Cas9-based OTUB1 knockout (KO) cells. B, U2OS cells were transfected with control or SMART pool siRNA against the OTUB1 gene. Whole cell lysates were then analyzed for the expression of the indicated proteins by immunoblotting. C, expression of siRNA-resistant FLAG-OTUB1 rescues UBE2E1 protein levels in OTUB1 knockdown cells. U2OS stable cell lines with control plasmid or FLAG-OTUB1^res were transfected with nontarget siRNA or individual siRNA against endogenous OTUB1 gene and assayed for the steady-state levels of the indicated proteins by immunoblotting. D, introduction of untagged OTUB1 or FLAG-OTUB1 in OTUB1 knockout U2OS cells rescues UBE2E1 levels. E, overexpression of OTUB1 enhances UBE2E1 proteins levels. U2OS WT cells were transiently transfected with either empty vector or FLAG-OTUB1 plasmid and whole cell lysate was analyzed by Western hybridization. F, WT FLAG-OTUB1^WT and catalytic mutant FLAG-OTUB1^C91S but not E2 interacting mutant FLAG-OTUB1^T134R rescued the steady-state levels of UBE2E1 in OTUB1 knockdown U2OS cells.

Figure 3.

OTUB1 rescues UBE2E1 from proteasomal degradation. A, cycloheximide chase to assess the half-life of UBE2E1 in control and OTUB1 knockdown U2OS cells. Expression of siRNA-resistant OTUB1 increases the half-life of UBE2E1 in OTUB1 knockdown cells. UBE2E1 (middle band) levels were normalized against the tubulin loading control and graphed on the bottom left (B). C, control and OTUB1 knockdown U2OS cells were treated with MG132 (10 μm) for 1- and 2-h time points and whole cell lysate was assayed for the steady-state levels of UBE2E1 by immunoblotting.

Figure 4.

OTUB1 suppresses autoubiquitination of UBE2E1. In vitro assay of recombinant proteins showing autoubiquitination of UBE2E1 in the presence and absence of the E3 ligase, RNF4, and OTUB1-C91S. Coomassie-stained gel of ubiquitination reactions containing 0.1 nm UBA1, 5 μm UBE2E1, 1 μm RNF4Δ22, 50 μm ubiquitin, and 10 μm OTUB1 C91S. Western blotting of reactions shown in top panel using antibodies against UBE2E1 (middle) and Lys-48–polyubiquitin (bottom).

Figure 5.

UBE2E1 is autoubiquitinated in U2OS cells. A, U2OS cells were co-transfected with plasmids expressing HA-UBE2E1 and His₆-ubiquitin. His₆-tagged ubiquitinated proteins in the whole cell lysate were enriched using Ni²⁺-NTA–agarose beads and analyzed by Western hybridization with anti-HA antibody. B, U2OS cells were co-transfected with plasmids expressing either WT HA-UBE2E1 or catalytically inactive HA-UBE2E1 (C131A) and His₆-ubiquitin. His₆-tagged ubiquitinated proteins and whole cell lysate were analyzed as described in A. Asterisk denotes cross-reactive bands. C and D, cycloheximide chase to assess the half-life of WT HA-UBE2E1^WT and catalytic mutant HAUBE2E1^C131A in U2OS cells.

Figure 6.

Depletion of OTUB1 or UBE2E1 does not affect global H2A-Ub and H2B-Ub levels. Whole cell lysates and acid-extracted histones were analyzed by immunoblotting with the indicated antibodies. A, control and OTUB1 knocked down U2OS cells. B, U2OS WT and CRISPR OTUB1 knockout cells. C, control and UBE2E1 knockdown U2OS cells.