Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

Choi, Yun‐Seok; Lee, Yun-Ju; Lee, Seo-Yeon; Shi, Lei; Ha, Jung-Hye; Cheong, Hae‐Kap; Cheong, Chaejoon; Cohen, Robert E.; Ryu, Kyoung‐Seok

doi:10.1074/jbc.m114.624809

Cited by 23 publications

(34 citation statements)

References 38 publications

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“…UBE2G1 and its paralog UBE2G2 share similar domain structures with CDC34. A common structural feature of these three E2 enzymes is an acidic loop (Figure S5A, highlighted with red) in the vicinity of their respective active site cysteines (Figure S5A, highlighted with blue), which facilitates the direct binding with ubiquitin and enables K48-linked ubiquitin chain assembly in the absence of associated E3 ligases (Choi et al, 2015). In association with gp78, an ER membrane bound RING finger E3 ubiquitin ligase, UBE2G2 directly tags misfolded proteins with K48-linked ubiquitin chains preassembled on the catalytic cysteine of UBE2G2, resulting in ER associated protein degradation (ERAD) (Li et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

“…This orchestrated action between UBE2D3 and UBE2G1 closely resembles the cooperativity of UBE2D3 and Cdc34 in promoting IκBα polyubiquitination mediated by SCF βTRCP2 (Wu et al, 2010), except that unlike Cdc34, UBE2G1 does not possess the ability to transfer ubiquitin onto substrates without prior ubiquitin conjugation. UBE2G1 was known to produce K48-linked poly-ubiquitin chains in the absence of an E3 ubiquitin ligase (Choi et al, 2015), but UBE2G1 cannot promote GSPT1 ubiquitination in the absence of CC-885 or Cul4A-Rbx1 (Figure 4E), indicating that close proximity of UBE2G1 to cereblon neomorphic substrates bridged by CRL4 CRBN is required to increase the processivity of UBE2G1 at physiological concentrations. This provides an explanation to how cereblon modulating agents can induce effective substrate degradation via K48-linked polyubiquitination, as well as speaks to the potential role of UBE2G1 in mediating the ubiquitination and degradation of other CRL4 cognate and/or neomophic substrates.…”

Section: Discussionmentioning

confidence: 99%

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UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

Lü¹,

Weng²,

Matyskiela³

et al. 2018

Preprint

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20The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide as well as 21 the novel cereblon modulating agents (CMs) including CC-122, CC-220 and cereblon-based 22 proteolysis-targeting chimaeras (PROTACs) repurpose the Cul4-RBX1-DDB1-CRBN 23 (CRL4 CRBN ) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic 24 substrates via polyubiquitination in conjunction with an E1 ubiquitin-activating enzyme and E2 25 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the 26 ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the 27 polyubiquitination of CRL4 CRBN neomorphic substrates in a cereblon-and CM-dependent 28 manner via a sequential ubiquitination mechanism: UBE2D3 transforms the neomorphic 29 substrates into mono-ubiquitinated forms, upon which UBE2G1 catalyzes K48-linked 30 polyubiquitin chain extension. Blockade of UBE2G1 diminishes the ubiquitination and 31 degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested 32CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma 33 survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring 34 drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent 35 IKZF1/3 degrader CC-220. Collectively, these findings suggest that loss of UBE2G1 activity 36 might be a resistance mechanism to drugs that hijack the CRL4 CRBN to eliminate disease-driving 37 proteins, and that this resistance mechanism can be overcome by next-generation CMs that 38 destroy the same targeted protein more effectively. 39 40 41 42The ubiquitin-proteasome system (UPS) is a highly regulated component of the protein 43 homeostasis network that dictates multiple cellular processes in eukaryotes (Hershko and 44 Ciechanover, 1998). Through the orchestrated actions of ubiquitin-activating enzymes (E1), 45 ubiquitin-conjugating enzymes (E2) and ubiquitin-ligating enzymes (E3), the ε-amine of a lysine 46 residue in a target protein is covalently conjugated with K48-or K11-linked poly-ubiquitin 47 chains, thereby marking the target protein for proteasomal degradation (Jin et al., 2008; 48 Komander and Rape, 2012; Pickart, 2001). Recently, repurposing the Cullin-Ring E3 ligase 49 complexes CRL4 CRBN (Cul4-RBX1-DDB1-CRBN) and CRL2 VHL (Cul2-RBX1-EloB/C-VHL) 50 with small-molecule degraders to remove disease-driving proteins otherwise considered 51 'undruggable' has emerged as a novel therapeutic modality that has the potential to transform 52 drug discovery and development (Bondeson and Crews, 2017; Huang and Dixit, 2016; Lebraud 53 and Heightman, 2017). 54There are two types of small-molecule degraders that have been exploited used to date. The first 55 is represented by thalidomide (THAL), lenalidomide (LEN) and pomalidome (POM), as well as 56 other cereblon modulating agents This class of molecule docks 57 into a tri-tryptophan pocket in the thalidomide...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

Lü¹,

Weng²,

Matyskiela³

et al. 2018

Preprint

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show abstract

“…In contrast to this well-known deciphering function of UBDs, the function of ubiquitin binding in polyubiquitin chain formation is poorly understood. For instance, the activities of the E2 enzymes Ubc13 (Pastushok et al, 2005), Ube2g1 (Choi et al, 2015), and Cdc34 (Choi et al, 2010) rely on ubiquitin binding events. In addition, it has already been shown that processive polyubiquitin chain formation can be promoted by noncovalent interactions of ubiquitin with the backside of certain E2 enzymes (Brzovic et al, 2006;Buetow et al, 2015) as well as by RING domains showing ubiquitin binding activity (Brown et al, 2014;Wright et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation

et al. 2016

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Ubiquitin conjugation is an essential process modulating protein function in eukaryotic cells. Surprisingly, little is known about how the progressive assembly of ubiquitin chains is managed by the responsible enzymes. Only recently has ubiquitin binding activity emerged as an important factor in chain formation. The Ubc7 activator Cue1 carries a ubiquitin binding CUE domain that substantially stimulates K48-linked polyubiquitination mediated by Ubc7. Our results from NMR-based analysis and in vitro ubiquitination reactions point out that two parameters accelerate ubiquitin chain assembly: the increasing number of CUE binding sites and the position of CUE binding within a growing chain. In particular, interactions with a ubiquitin moiety adjacent to the acceptor ubiquitin facilitate chain elongation. These data indicate a mechanism for ubiquitin binding in which Cue1 positions Ubc7 and the distal acceptor ubiquitin for rapid polyubiquitination. Disrupting this mechanism results in dysfunction of the ERAD pathway by a delayed turnover of substrates.

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“…Ube2R1/2 is a critical E2 that functions with the cullin-RING ubiquitin ligases (1,18), and, together, these enzymes may be responsible for 20% of all proteasome-dependent degradation in human cells (19). Ube2R1/2 contains an atypical insertion distal to its active site that contains several conserved acidic residues (20)(21)(22)(23)(24)(25), and this acidic loop has also been shown to have an important role in Lys 48 selectivity (26,27). More recent work has identified a loop on acceptor ubiquitin that contains residues that interact with the E2 in order to help place Lys 48 in the E2 active site (26).…”

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confidence: 99%

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

Hill

Harrison

Lewis

et al. 2016

Molecular and Cellular Biology

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c Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and Asp 143 on Ube2R1/2. The validity of this interaction was confirmed through biochemical experiments. Since structural examples involving Arg 54 in protein-ubiquitin complexes are exceedingly rare, these results provide additional insight into how ubiquitin-protein complexes can be stabilized. We discuss how these findings relate to how other ubiquitin-conjugating enzymes direct the lysine specificity of polyubiquitin chains.

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Differential Ubiquitin Binding by the Acidic Loops of Ube2g1 and Ube2r1 Enzymes Distinguishes Their Lys-48-ubiquitylation Activities

Cited by 23 publications

References 38 publications

UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation

Mechanism of Lysine 48 Selectivity during Polyubiquitin Chain Formation by the Ube2R1/2 Ubiquitin-Conjugating Enzyme

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