UBE2S Learns Self-Control.

SUMMARY

In this issue of Structure, Liess et al. (2019) demonstrate that the cell cycle regulator UBE2S shuts itself off through autoubiquitination at a conserved lysine residue. Since E2s are at the center of the ubiquitination cascade, this presents a possible regulatory mechanism in a multitude of cellular processes.

PREVIEW

Ubiquitination is a significant and dynamic form of eukaryotic protein regulation that governs a wide range of cellular processes including cell cycle regulation, DNA repair, and immune response. During the cell cycle, substrate ubiquitination needs to be carried out at the right time and place to ensure the correct ordering of cellular events to avoid homeostatic imbalance. Precise regulation is achieved through the control of two E1, more than thirty E2, and more than six hundred E3 enzymes (Buetow and Huang, 2016). Lorenz and coworkers demonstrate that the cell cycle regulator UBE2S contains a self-inactivation mechanism that may be applicable to nearly a quarter of the E2s (Liess et al., 2019).

Substrate ubiquitination cannot occur haphazardly and errant regulation of ubiquitination results in numerous diseases including cancer (Rape, 2018). Within the overall ubiquitination cascade, E2s are the intermediaries, shuttling ubiquitin (Ub) from the E1 and working with the E3 to ubiquitinate target substrates. RING E3s often promote an activated, closed conformation of E2~Ub (~denotes covalent) to facilitate the transfer of Ub from the E2 to an E3-bound substrate. Regulation of this system can occur at multiple levels in the cascade, e.g. phosphorylation or conjugation of other ubiquitin-like proteins such as SUMO and NEDD8 (Buetow and Huang, 2016). However, understanding the regulation of E3s has largely been the focus and despite their significance, relatively little is known about the structural mechanisms of E2 regulation.

The gigantic, multisubunit 1.2 MDa E3 ligase known as the Anaphase-Promoting Complex/Cyclosome (APC/C) has a well-established role as the gatekeeper of the cell cycle (Rape, 2018). APC/C ubiquitinates its targets with the assistance of two E2s, UBE2C (UBCH10) and UBE2S, to mark cell cycle proteins with polyubiquitin chains for proteasomal destruction. APC/C recruits and activates UBE2C to initiate substrate polyubiquitination and UBE2S to extend polyubiquitin chains using distinct architectures and surfaces. In brief, UBE2C is recruited by the APC2 WHB and Ub transfer from UBE2C to substrates is facilitated by the canonical E2~Ub binding site on the APC11 RING. UBE2S uses an unprecedented mechanism where the catalytic ubiquitin-conjugating (UBC) domain of UBE2S binds to another surface of APC2 and the C-terminal extension docks to a groove composed of the APC2 and APC4 subunits. UBE2S~Ub does not need a RING to adopt the active, closed conformation, but rather the APC11 RING positions the substrate-linked Ub to receive another Ub from UBE2S to form a Lys11-linked chain (Brown et al., 2016; Wickliffe et al., 2011).

Since these enzymes are important for cell cycle timing, many E2s (including UBE2S) and their corresponding E3s are subject to delicate regulation. Both indirect and direct mechanisms regulating UBE2S function are known to exist, including phosphorylation and proteasomal degradation (Bremm et al., 2010; Craney et al., 2016; Williamson et al., 2009). The latter is postulated to occur through autoubiquitination of a lysine-rich C-terminal extension. In this issue of Structure, Lorenz and colleagues demonstrate that regulation of UBE2S and other E2s deserves more attention as autoubiquitination of the UBC domain results in autoinhibition but not proteasomal degradation suggesting a dynamic and responsive self-regulatory mechanism (Liess et al., 2019).

During ubiquitination reactions, autoubiquitination of the E2 is readily observed, especially in the absence of an E3. This poses a challenge for the full mechanistic characterization of the enzyme as it is often unclear whether this autoubiquitination event is relevant or a side product of reconstituted reactions. To tackle this problem, Liess et al. use a suite of methodologies, including in vitro ubiquitination assays, molecular dynamics simulations, NMR, quantitative mass spectrometry, immunoprecipitation, and cell-based assays, to investigate the significance of a specific autoubiquitination event on UBE2S (Liess et al., 2019).

The C-terminal extension of UBE2S has been ascribed to be its primary regulatory ubiquitination site. However, these are not the only lysines targeted. Interestingly, a sequence alignment of 34 human E2s, including UBE2S, reveals that a lysine near the active site cysteine (Lys⁺⁵) is present in ~25% of E2s. Solving additional crystal structures of the catalytic core of UBE2S (UBE2S^UBC) revealed flexibility within the active site of UBE2S^UBC. Specifically, Lys⁺⁵ (Lys100) was positioned in either a Lys⁺⁵-in or a Lys⁺⁵-out state where the primary amine of lysine is close to or moved away from the catalytic cysteine (Cys95), respectively. Molecular dynamics simulations demonstrated that the Lys-out state is energetically favored. However, enzyme assays with active and inactive versions of UBE2S^UBC show that the ubiquitination of this particular lysine residue occurs through intramolecular transfer supporting the Lys⁺⁵-in state and active site flexibility (Figure 1A).

Figure 1. Schematic of UBE2S self-inactivation mechanisms and implications.

(A) UBE2S can exist in Lys⁺⁵-in or Lys⁺⁵-out states. The Lys⁺⁵-in conformation brings Lys⁺⁵ (Lys100) in proximity to the active site Cys (C^cat, Cys95) for the intramolecular transfer of Ub. This mechanism is postulated to be conserved in potentially ~25% of all E2s.

(B) UBE2S can transfer its donor Ub from an active, closed UBE2S~Ub to 1) Lys11 on a substrate-linked Ub to form chains on E3-bound substrates for proteasomal degradation; 2) Lys⁺⁵ inactivating UBE2S by blocking E1-mediated recharging of UBE2S-Ub with another donor Ub; or 3) lysines on the C-terminal peptide (CTP) extension of UBE2S marking itself for destruction.

To understand the impact of autoubiquitination on enzymatic function, the monoubiquitinated form with a Ub molecule conjugated at Lys⁺⁵ of UBE2S, referred to as UBE2S-Ub, was purified, validated by mass spectrometry, and used to assay the different roles of UBE2S in the E1-E2-E3 cascade. In summary, it was found that UBE2S-Ub could not readily accept another Ub from the E1 or form Lys11-linked Ub chains. Furthermore, the authors found that Ube2S-Ub adopts a conformation similar to the active, closed conformation by NMR. However, using in vitro enzyme assays with APC/C and full-length UBE2S, the autoubiquitination pattern and the enzymatic activity was nearly identical between the wild-type and the K⁺⁵R variant of UBE2S, suggesting a significant portion of UBE2S autoubiquitination occurs on the C-terminal extension.

A critical question remained: what is the functional significance of this autoubiquitination of UBE2S at Lys⁺⁵ in the cell? After generating a polyclonal antibody for UBE2S that can monitor both free and ubiquitinated forms of UBE2S and performing co-immunoprecipitations, the authors found that a significant portion of UBE2S exists in a monoubiquitinated form. Tandem mass tag mass spectrometry then revealed the existence of UBE2S automodified at Lys⁺⁵ suggesting a role for autoinhibition in cells. Intriguingly, total amounts of UBE2S and UBE2S-Ub decreased during mitotic exit. UBE2S decreased because of proteasomal destruction independent of the automodification of UBE2S at Lys⁺⁵ (Figure 1B). Taken together, these results imply that the self-inactivation mechanism is cell cycle regulated.

The authors propose the presence of a potential deubiquitinase (DUB) that specifically removes these Ub marks from UBE2S and possibly other E2s. Similar to kinases and phosphatases, DUBs counteract the activity of E2s and E3s, including Lys11-linkages formed by APC/C-UBE2S (Bonacci et al., 2018). Since this Lys⁺⁵-linked Ub tag is not for proteasomal-mediated UBE2S destruction, one or multiple DUBs could assist in controlling ubiquitination at an earlier step in the cascade than previously thought.

This study leaves us with several interesting questions to be answered in the future. First, what cellular factors, e.g. DUBs and APC/C accessibility, regulate UBE2S autoubiquitination at Lys⁺⁵ and the C-terminus? Interestingly, the highest levels of UBE2S autoinhibition was observed when APC/C function is restrained in mitosis. Second, how is the cell cycle regulated by Lys⁺⁵ automodification? Both APC/C E2s, UBE2C and UBE2S, have Lys⁺⁵ conserved across several organisms, and thus would be susceptible to self-regulation.

More broadly, the conservation of this lysine across the E2 family suggests that controlling autoinhibition is relevant to many cellular processes. Indeed, this self-inactivation mechanism was already observed for UBE2T, an E2 vital to the Fanconi anemia pathway for DNA repair (Machida et al., 2006). Furthermore, the creation of autoinhibited E2-Ubs would change the pool of available E2s to cooperate with E3s. As E2s are in the middle of the cascade and multiple E3s use multiple E2s, the regulatory effects in downstream signaling could then be propagated rapidly and widely.

E2s and E3s are susceptible to dynamic changes by post-translational modifications and protein-protein interactions. Self-regulation, autoinhibition by autoubiquitination, is yet another layer of complexity to an already complicated process. Nevertheless, Liess et al. (2019) provide a strong foundation for future work that is needed to fully appreciate the significance of this regulatory mechanism.