微纳米DNA探针捕获蛋白质-质谱分析联用研究肿瘤细胞对金属抗肿瘤药物的应答机制

Cytotoxic metal-based anticancer drugs, such as cisplatin and its analogs, attack and damage DNA, which subsequently induces apoptosis or necrosis by blocking the replication and transcription of DNA. However, there is not a direct and simple relationship between the amount of DNA-drug adducts formed in different types of cancer cells and the cytoxicity of the drugs for respective cancer cells, and some types of cancer cells can adequately repair or bypass the drug-damaged DNA so that the cancer cells become resistant to drugs. This implies that different types of cancer cells possess different mechanisms responding for DNA lesions made by various anticancer drugs, which are determined by the recognition and repairing capacity of the key regulatory proteins for DNA lesions in cancer cells. However, it is still largely unclear about the recognition and repairing mechanism of regulatory proteins' response for DNA lesions. To address this key scientific issue, the present project aims developing novel methodology to capture and identify proteins which recognize DNA lesions made by various anticancer drugs. Firstly, we will design and construct drug-damaged DNA probes by immobilized DNA-drug adducts on the surface of magnetic beads or gold nanoparticles, and these probes are utilized to capture regulatory proteins in the lysates of different types of cancer cells, the captured proteins are then identified by mass spectrometry (MS). Secondly, combined with molecular modeling, hydrogen/deuterium exchange MS (HX-MS) developed previously in our lab will be applied to elucidate the binding modes and sites of proteins with DNA-drug adducts. The proposed research is expected to provide novel insights into the molecular mechanism of cancer cells responding to drug-made lesions of DNA, which will be very helpful to design and develop novel anticancer drugs.

顺铂等细胞毒性金属抗癌药物通过造成DNA损伤，阻止DNA转录和复制，诱导细胞凋亡或坏死。但是，细胞内药物与DNA形成复合物的量与其细胞毒性并不是简单的正比关系，某些癌变细胞可修复或逃避化疗药物引起的DNA损伤,对化疗产生抗药性，意味着不同肿瘤细胞对DNA的药物损伤有不同的应答机制。然而，有关肿瘤细胞如何识别、应答化疗药物造成的DNA损伤仍然不清楚。本项目将针对肿瘤细胞应答DNA药物损伤的分子机制这一关键科学问题，构建微纳尺寸的DNA探针，捕获、富集不同类型肿瘤细胞中识别药物损伤DNA的调控蛋白，建立基于质谱的蛋白质组学研究方法，鉴定捕获的蛋白质组；进而应用我们前期建立的氢氘交换质谱分析方法，结合分子模拟技术，分析鉴定关键调控蛋白和DNA－药物复合物的作用模式和位点，为深入理解肿瘤细胞对DNA药物损伤的分子应答机制，优化金属抗肿瘤药物的分子设计提供理论依据。

顺铂等细胞毒性金属抗癌药物通过造成DNA 损伤，阻止DNA 转录和复制，诱导细胞凋亡或坏死。但是，细胞内药物与DNA 形成复合物的量与其细胞毒性并不是简单的正比关系，某些癌变细胞可修复或逃避化疗药物引起的DNA 损伤,对化疗产生抗药性，意味着不同肿瘤细胞对DNA 的药物损伤有不同的应答机制。然而，有关肿瘤细胞如何识别、应答化疗药物造成的DNA 损伤仍然不清楚。本项目将针对肿瘤细胞应答DNA 药物损伤的分子机制这一关键科学问题，通过四年的研究与探索，借助构建的微纳尺寸的DNA 亲和探针，实现在生理条件下灵敏捕获并鉴定肿瘤细胞中识别铂类药物损伤DNA的调控蛋白，筛选出了特异性识别trans-PtTz-1,3-链内交联DNA的染色体相关蛋白PC4和RFA1。采用氢氘交换质谱分析方法，结合计算机模拟分子动力学实验及凝胶阻滞的实验，研究了trans-PtTz-1,3-链内交联DNA复合物与PC4蛋白的非共价相互作用。确定了结合位点位及结合会使PC4蛋白二聚化结构的影响，确定了PC4蛋白上的俩个氨基酸残基86位精氨酸与104位丝氨酸对PC4与损伤DNA的相互识别中起到重要的作用。整个项目工作取得了预期的研究成果，且为下一步捕获并鉴定出特异性识别与其它药物损伤DNA的结合蛋白，更深入研究DNA结合蛋白HMGB1、p53等和DNA-金属抗肿瘤药物复合物的非共价键弱相互作用，阐明DNA结合蛋白在细胞药物应答中的功能和作用奠定了坚实的方法学基础。