Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, MN, 55455, USA.
Center for Genomic Integrity, Institute for Basic Science, Ulsan, Republic of Korea.
DNA Repair (Amst). 2020 May;89:102840. doi: 10.1016/j.dnarep.2020.102840. Epub 2020 Mar 19.
1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu, Lys, Cys, His, Arg, and Cys. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH){d(GpG)}] cross-links which were detected by HPLC-ESI-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.
1,1,2,2-顺式二氨二氯铂（II）（顺铂）是一种广泛应用于临床治疗各种癌症的化疗药物。顺铂的抗肿瘤活性通常归因于其通过顺式铂化 DNA 双螺旋中的两个亲核位点，形成链内和链间 DNA-DNA 交联的能力。然而，顺铂还会诱导 DNA-蛋白质损伤（DPCs），由于其能够阻断 DNA 复制和转录，这些损伤可能对其生物学效应有贡献。我们之前报道过，在顺铂处理的人 HT1080 细胞中，超过 250 种核蛋白，包括高迁移率族蛋白、组蛋白和延伸因子，形成了 DPCs（Ming 等人，Chem. Res. Toxicol. 2017, 30, 980-995）。有趣的是，顺铂诱导的 DNA-蛋白质缀合物在加热时通过未知机制逆转。在本工作中，我们使用 DNA 修复蛋白 O-烷基鸟嘌呤 DNA 烷基转移酶（AGT）作为模型，研究顺铂介导的 DNA-蛋白质交联的分子细节，并建立其逆转的机制。我们发现，在顺铂存在的情况下，AGT 很容易与 DNA 发生交联。来源于 dG-Pt-AGT 复合物的胰蛋白酶肽的 HPLC-ESI-MS/MS 测序表明，交联发生在该蛋白的六个位点，包括 Glu、Lys、Cys、His、Arg 和 Cys。通过与相应的标准品相比，对修饰蛋白的总消化产物进行 HPLC-ESI-MS/MS 分析，检测到顺铂诱导的 Lys-Gua 交联（1,1-顺式二胺-2-（5-氨基-5-羧基戊基）氨基-2-（2'-脱氧鸟苷-7-基）-铂（II）（dG-Pt-Lys）。加热时，dG-Pt-AGT 复合物发生从蛋白质到 DNA 的顺式铂迁移，形成 cis-[Pt(NH){d(GpG)}] 交联，通过 HPLC-ESI-MS/MS 检测到。我们的结果为顺铂介导的 DNA-蛋白质交联及其与相应的 DNA-DNA 损伤的动态平衡的机制提供了新的见解。
