A novel ubiquitin ligase is deficient in Fanconi anemia

Meetei, Amom Ruhikanta; Winter, Johan P. de; Medhurst, Annette L.; Wallisch, Michael; Waisfisz, Quinten; Vrugt, Henri J. van de; Oostra, Anneke B.; Yan, Zhijiang; Chen, Ling; Bishop, Colin E.; Hoatlin, Maureen E.; Joenje, Hans; Wang, Weidong

doi:10.1038/ng1241

Cited by 533 publications

(581 citation statements)

References 29 publications

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“…In the case of FancD2, carboplatin led to the appearance of a second protein species with higher molecular weight ('FancD2-L'; Figure 6a), corresponding to monoubiquitinated FancD2 that is induced by the upstream components of the pathway upon activation. 31 When carboplatin was combined with ganetespib, FancD2 was not only reduced in its total levels, but the upper band corresponding to ubiquitinated FancD2 disappeared completely. Thus, both the key components and key functionality of the FA pathway are compromised by HSP90 inhibition.…”

Section: Carbomentioning

confidence: 93%

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

et al. 2016

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All current regimens for treating ovarian cancer center around carboplatin as standard first line. The HSP90 inhibitor ganetespib is currently being assessed in advanced clinical oncology trials. Thus, we tested the combined effects of ganetespib and carboplatin on a panel of 15 human ovarian cancer lines. Strikingly, the two drugs strongly synergized in cytotoxicity in tumor cells lacking wild-type p53. Mechanistically, ganetespib and carboplatin in combination, but not individually, induced persistent DNA damage causing massive global chromosome fragmentation. Live-cell microscopy revealed chromosome fragmentation occurring to a dramatic degree when cells condensed their chromatin in preparation for mitosis, followed by cell death in mitosis or upon aberrant exit from mitosis. HSP90 inhibition caused the rapid decay of key components of the Fanconi anemia pathway required for repair of carboplatin-induced interstrand crosslinks (ICLs), as well as of cell cycle checkpoint mediators. Overexpressing FancA rescued the DNA damage induced by the drug combination, indicating that FancA is indeed a key client of Hsp90 that enables cancer cell survival in the presence of ICLs. Conversely, depletion of nuclease DNA2 prevented chromosomal fragmentation, pointing to an imbalance of defective repair in the face of uncontrolled nuclease activity as mechanistic basis for the observed premitotic DNA fragmentation. Importantly, the drug combination induced robust antitumor activity in xenograft models, again accompanied with depletion of FancA. In sum, our findings indicate that ganetespib strongly potentiates the antitumor efficacy of carboplatin by causing combined inhibition of DNA repair and cell cycle control mechanisms, thus triggering global chromosome disruption, aberrant mitosis and cell death. 6 This provides a strong rationale for HSP90 inhibitors in cancer therapy 7 and raises the possibility that HSP90 inhibitors can be used in combination with DNAdamaging chemotherapeutics. 8 Ovarian cancer is among the most common gynecological tumor types and carries the worst prognosis. HSP90 is highly expressed in advanced ovarian carcinoma 9 and thus constitutes a potential therapeutic drug target. 10 In support, HSP90 inhibitors were found effective against ovarian cancer in preclinical models, 11-14 alone or in combination with conventional chemotherapy. 15,16 Carboplatin is the key component of all current first-line therapies for ovarian carcinoma. However, while initially often responding with regression, most tumors relapse and develop resistance within less than a year. 17,18 Like other platinum compounds, carboplatin acts by forming crosslinks within DNA. 17 Platinum compounds form intrastrand DNA lesions but also interstrand crosslinks (ICLs) by covalently linking opposite DNA strands. Such ICLs represent major obstacles to the DNA replication fork 19 and are therefore considered the principal toxic lesion of carboplatin's mode of action. The ICLs can only be removed by a sophisticated DNA repair systemthe Fanconi ...

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Section: Carbomentioning

confidence: 93%

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

et al. 2016

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“…2d), suggesting that RAD18 is not the E3 ligase for USP1 degradation. Previous studies have indicated that the UV-dependent monoubiquitination of FANCD2 requires an intact Fanconi anaemia core complex 20,21 -a multisubunit E3 ligase containing at least eight Fanconi anaemia protein subunits (A, B, C, E, F, G, L and M) 22,23 . The Fanconi anaemia core complex was systematically excluded from the degradation of USP1, as analysis of patient-derived Fanconi anaemia fibroblasts from different Fanconi anaemia subtypes (FA-A, FA-C, FA-G and FA-L) showed that they remained competent for USP1 degradation and for the monoubiquitination of PCNA (Fig.…”

Section: Uv Damage Degrades Usp1 and Increases Pcna Monoubiquitinationmentioning

confidence: 99%

Regulation of monoubiquitinated PCNA by DUB autocleavage

et al. 2006

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Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.Monoubiquitination is a highly regulated process that is conserved in all eukaryotes 1,2 and controls a broad range of cellular functions, including DNA repair. Protein monoubiquitination is a reversible post-translational event that can be influenced by the opposing activities of a ubiquitin E3 ligase and a deubiquitinating enzyme (DUB), similar to the regulation of protein phosphorylation by kinases and phosphatases 3,4 . Protein monoubiquitination regulates the rescue of stalled DNA replication forks -an important cellular process required for cell survival 5 . The E2 ubiquitin conjugating enzyme RAD6 and the E3 ligase RAD18 are conserved in both yeast and human and they coordinate and activate the monoubiquitination of PCNA in response to UV damage or stalled replication forks [6][7][8] . Recent studies have shown that polη, a specialized TLS polymerase, is recruited to the replication fork through a specific interaction with monoubiquitinated PCNA 7 . The deployment of TLS polymerases during replication ensures timely bypass of the diverse DNA lesions encountered by the replication fork. Although many TLS polymerases are intrinsically mutagenic, polη allows replication past UV-damaged bases with high fidelity 9-11 . Thus, a model has emerged in which polη binds to monoubiquitinated PCNA and ensures accurate (error-free) replicative bypass of UV lesions. However, other (error-prone) TLS polymerases (such as polι and Rev1) have recently been shown to rely on monoubiquitinated PCNA for their function 12,13 . How cells limit PCNA monoubiquitination and the unwanted deployment of polη and/or other error-prone TLS polymerases in the absence or presence of extrinsic DNA damage during the synthesis of DNA in S phase is not known.In humans, protein deubiquitination is controlled by a family of approximately 95 distinct DUB enzymes 14,15 but the function of most of these proteins is unknown. DUBs are cysteine proteases that cleave ubiquitin fro...

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“…We showed that FAAP43 (also called FANCL) is a ubiquitin ligase and was defective in an individual affected with Fanconi anemia belonging to a new complementation group (FA-L) 3,7 (Fig. 1a).…”

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confidence: 99%