Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting

Hodge, Curtis D.; Ra, Edwards; Markin, Craig J.; McDonald, Darin; Pulvino, Msy; Huen, Msy; Zhao, Jinjing; Spyracopoulos, Leo; Hendzel, Michael J.; Glover, J. N. Mark

doi:10.1021/acschembio.5b00222

Cited by 57 publications

(63 citation statements)

References 50 publications

(113 reference statements)

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“…The nitro to cyano functional group swap was the main factor in markedly improving plasma stability. Nitro groups in similar compounds have been shown to cause unwanted irreversible covalent binding to proteins via a nucleophilic aromatic substitution reaction [87].…”

Section: Usp7mentioning

confidence: 99%

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

Kemp¹

2016

Progress in Medicinal Chemistry

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Section: Usp7mentioning

confidence: 99%

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

Kemp¹

2016

Progress in Medicinal Chemistry

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“…As such, targeting the enzymes responsible for either the addition or removal of ubiquitin offers a potential therapeutic avenue. Encouragingly, a number of small molecule inhibitors of ubiquitin-associated enzymes are currently entering pre-clinical trials (51)(52)(53)(54)(55). Here, we screened the TCGA cervical cancer database for the expression of DUBs, enzymes which catalyse the remove of ubiquitin from protein substrates (5).…”

Section: Discussionmentioning

confidence: 99%

The deubiquitinase (DUB) USP13 promotes Mcl-1 stabilisation in cervical cancer

Morgan

Patterson

Barba-Moreno

et al. 2020

Preprint

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Ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in approximately 15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic Bcl-2 family and the reduced Mcl-1 expression contributed to the observed proliferative defect. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

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“…Ubiquitination Assay-All ubiquitination assays were done essentially as described previously (47). The reactions were run for 15, 30, 45, and 90 min at 37°C and quenched with SDS-PAGE loading buffer, and the results were visualized by Western blotting.…”

Section: Methodsmentioning

confidence: 99%

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

Hodge

Ismail

Edwards

et al. 2016

Journal of Biological Chemistry

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DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

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Covalent Inhibition of Ubc13 Affects Ubiquitin Signaling and Reveals Active Site Elements Important for Targeting

Cited by 57 publications

References 50 publications

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

Recent Advances in the Discovery of Deubiquitinating Enzyme Inhibitors

The deubiquitinase (DUB) USP13 promotes Mcl-1 stabilisation in cervical cancer

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

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