Ubiquitin Chain Elongation Requires E3-Dependent Tracking of the Emerging Conjugate

Kelly, Aileen; Wickliffe, Katherine E.; Song, Ling; Fedrigo, Indro; Rapé, Michael

doi:10.1016/j.molcel.2014.09.010

Cited by 69 publications

(145 citation statements)

References 48 publications

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“…Recently, two studies dissected the detailed mechanism used by Ube2s and its E3 APC to drive polyubiquitin chain formation on substrates. 54,55 In their model, APC simultaneously associates with Ube2s and substrate, and continuously adds Ub to the distal end of the growing polyubiquitin chain in a one-by-one manner. We speculate that the high affinity between Ube2s and Sox2 may enhance the efficiency of polyubiquitin chain formation on Sox2.…”

Section: Discussionmentioning

confidence: 99%

Ube2s regulates Sox2 stability and mouse ES cell maintenance

et al. 2015

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Sox2 has a critical role in embryonic stem (ES) cell maintenance and differentiation. Interestingly, its activity is highly dosagedependent. Although transcriptional regulation of Sox2 has been extensively studied, the mechanisms orchestrating its degradation remain unclear. In this study, we identified ubiquitin-conjugating enzyme E2S (Ube2s) as a novel effector for Sox2 protein degradation. Ube2s mediates K11-linked polyubiquitin chain formation at the Sox2-K123 residue, thus marking it for proteasome-mediated degradation. Besides its role in fine-tuning the precise level of Sox2, Ube2s reinforces the self-renewing and pluripotent state of ES cells. Importantly, it also represses Sox2-mediated ES cell differentiation toward the neural ectodermal lineage.

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Section: Discussionmentioning

confidence: 99%

Ube2s regulates Sox2 stability and mouse ES cell maintenance

et al. 2015

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“…This LR tail-binding site does not change conformation on interconversion between active and inactive states (12,39). Although independent of the catalytic module for APC/C binding, the catalytic activity of Ube2S requires the RING domain of Apc11 that is repurposed to engage the acceptor ubiquitin of the APC/C substrate for covalent linkage with the donor ubiquitin of the Ube2S-ubiquitin conjugate (14,30). The acceptor ubiquitin-binding site on Apc11 is accessible in the inactive APC/C conformation ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The work raises the possibility that conformational changes of the Apc1 WD40 domain may play a role in regulating UbcH10 binding to the APC/C. position the acceptor ubiquitin, conjugated to an APC/C substrate, for modification by Ube2S (14,30,31). UbcH10 and Ube2S together build branched ubiquitin chains on APC/C substrates, and these chains are recognized more efficiently by the proteasome (32).…”

Section: Significancementioning

confidence: 99%

“…APC/C activity is controlled by its association with one of two coactivator subunits (either Cdc20 or Cdh1) that function to specify substrate recognition and stimulate ubiquitin transfer reactions (11)(12)(13)(14). The mitotic coactivator Cdc20 preferentially binds to hyperphosphorylated APC/C, whereas Cdh1 also binds to unphosphorylated APC/C.…”

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confidence: 99%

“…The association of UbcH10 with the RING domain of Apc11 (Apc11 RING ) and Apc2's winged-helix B domain (Apc2 WHB ) initiates ubiquitin-chain formation (19,20,(24)(25)(26). Ube2S, on the other hand, is responsible for ubiquitin-chain extension and specifically assembles Lys11-linked ubiquitin chains on substrates targeted by the APC/C (14,25,(27)(28)(29)(30). In vertebrates, the RING domain of Apc11 is repurposed to…”

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confidence: 99%

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WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

Chang

Aibara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

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Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

Song,

Mikami,

Majima

et al. 2024

Helvetica Chimica Acta

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Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i.e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

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Ubiquitin Chain Elongation Requires E3-Dependent Tracking of the Emerging Conjugate

Cited by 69 publications

References 48 publications

Ube2s regulates Sox2 stability and mouse ES cell maintenance

Ube2s regulates Sox2 stability and mouse ES cell maintenance

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

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