Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards.
Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards.
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抗肿瘤氮的DNA-蛋白交联的蛋白质组学分析。
DOI：
10.1021/tx900078y
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发表时间：
2009-06
影响因子：
4.1
通讯作者：
Tretyakova, Natalia Y.
中科院分区：
文献类型：
--
作者：
Loeber, Rachel L.;Michaelson-Richie, Erin D.;Codreanu, Simona G.;Liebler, Daniel C.;Campbell, Colin R.;Tretyakova, Natalia Y.
氮芥是临床上用于治疗各种肿瘤病症的抗肿瘤剂。这些化合物的生物活性通常归因于它们诱导DNA-DNA交联的能力。然而，氮MUR能够产生各种其他病变，包括DNA-蛋白质交联（DPC）。氮芥诱导的DPC没有得到很好的表征，因为它们的结构复杂性和以前可用的实验方法的特异性和灵敏度不足。在目前的工作中，亲和捕获方法结合质谱为基础的蛋白质组学，以确定哺乳动物蛋白质形成共价交联的DNA在一个简单的氮芥，氮芥的存在下。将5′-生物素化的DNA双链体与核蛋白提取物孵育后，通过链霉亲和素珠亲和捕获法分离DPC，并通过胰蛋白酶肽的HPLC-ESI+-MS/MS鉴定交联蛋白。氮芥处理导致DPC的形成，其中核蛋白参与染色质调节、DNA复制和修复、细胞周期控制、转录调节和细胞结构。蛋白质印迹分析用于确认蛋白质鉴定并定量药物介导的交联程度。在总蛋白水解酶中发现的氨基酸-核碱基缀合物的质谱分析显示，氮芥诱导的DPC是通过双链体DNA中鸟嘌呤的N7位置和蛋白质内的半胱氨酸硫醇的烷基化形成的，以得到N-[2-[S-半胱氨酰]乙基]-N-[2-（关-7-基）乙基]甲胺损伤。本文所述的结果表明，细胞暴露于氮霉素导致大量核蛋白与染色体DNA交联，可能导致这些药物的细胞毒性和致突变作用。
Nitrogen mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA-DNA cross-links. However, nitrogen mustards are able to produce a variety of other lesions, including DNA-protein cross-links (DPCs). DPCs induced by nitrogen mustards are not well characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple nitrogen mustard, mechlorethamine. Following incubation of 5′-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by HPLC-ESI+-MS/MS of tryptic peptides. Mechlorethamine treatment resulted in the formation of DPCs with nuclear proteins involved in chromatin regulation, DNA replication and repair, cell cycle control, transcriptional regulation, and cell architecture. Western blot analysis was employed to confirm protein identification and to quantify the extent of drug-mediated cross-linking. Mass spectrometry of amino acid-nucleobase conjugates found in total proteolytic digests revealed that mechlorethamine-induced DPCs are formed via alkylation of the N7 position of guanine in duplex DNA and cysteine thiols within the proteins to give N-[2-[S-cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]methylamine lesions. The results described herein suggest that cellular exposure to nitrogen mustards leads to cross-linking of a large spectrum of nuclear proteins to chromosomal DNA, potentially contributing to the cytotoxic and mutagenic effects of these drugs.
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O6-烷基鸟嘌呤DNA-烷基转移酶在人肝和黑色素瘤中的细胞内和细胞内分布的免疫组织学检查。
影响因子：
8.8
作者：
Lee, S M;Rafferty, J A;Elder, R H;Fan, C Y;Bromley, M;Harris, M;Thatcher, N;Potter, P M;Altermatt, H J;Perinat-Frey, T
通讯作者：
Perinat-Frey, T
影响因子：
4.1
作者：
Balcome, S;Park, S;Tretyakova, N
通讯作者：
Tretyakova, N
影响因子：
3
作者：
Belanich, M;Randall, T;Yarosh, DB
通讯作者：
Yarosh, DB
影响因子：
16.8
作者：
Daniels, DS;Woo, TT;Tainer, JA
通讯作者：
Tainer, JA
影响因子：
4.8
作者：
Barker, S;Weinfeld, M;Murray, D
通讯作者：
Murray, D