Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97.
Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97.
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DOI：
10.1080/15384101.2020.1860559
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Wani AA
中科院分区：
文献类型：
--
作者：
Chauhan AK;Li P;Sun Y;Wani G;Zhu Q;Wani AA
盐皮质激素和雄激素受体拮抗剂螺内酯最近被确定为核苷酸切除修复(NER)的抑制剂，其作用机制是通过诱导TFIIH成分着色性干皮病B蛋白(XPB)的蛋白分解起作用。这一活动为将螺内酯用于癌症治疗提供了强有力的理由。在这里，我们报道了螺内酯诱导的XPB蛋白分解是通过泛素选择性分离酶Valosin-Holding Protein(VCP)/p97介导的。我们发现螺内酯诱导XPB的降解呈剂量和时间依赖性，但不能诱导XPD的降解，并且VCP/p97抑制剂DBeQ、NMS-873和代谢抑制剂MLN4924抑制了XPB的降解。此外，VCP/p97抑制剂处理细胞可导致XPB泛素结合物的积聚，但不能积聚XPD。可诱导的shRNA敲除VCP/p97不影响XPB水平，但可抑制螺内酯诱导的XPB降解。此外，VCP/p97在螺内酯和蛋白酶体抑制剂MG132处理后与XPB相互作用，而VCP/p97接头UBXD7结合XPB及其泛素结合物。此外，ATP类似物介导的CDK7抑制显著减缓了螺内酯诱导的XPB的降解。同样，通过紫外线照射使TFIIH与NER结合可以减缓螺内酯诱导的XPB的降解。这些结果表明，螺内酯诱导的XPB蛋白降解需要VCP/p97功能，而HALO-TFIIH中的XPB比CORE-TFIIH中的XPB更容易被螺内酯诱导的蛋白降解。缩写NER：核苷酸切除修复；TFIIH：转录因子II H；CAK：CDK激活酶(CAK)复合体；XPB：B型着色性干皮病；VCP/p97：含有Valosin的蛋白/p97；CDK7：细胞周期蛋白依赖蛋白7；NAE：NEDD8激活酶；IP：免疫沉淀。
Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. Abbreviations NER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation
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