【作者】 赵林泓；

【导师】 刘扬中；

【作者基本信息】 中国科学技术大学 ， 生物无机化学， 2013， 博士

【摘要】 铂类和亚砷类药物是无机抗肿瘤药物中十分重要和典型的两类。铂和亚砷药物的生理学作用机制都与含硫蛋白质的相互作用密切相关。在这些蛋白中,铜转运蛋白在铂类药物的细胞摄取、转运、排出以及药效上发挥重要作用,而锌指蛋白则是亚砷化合物的极为重要的直接靶点。本论文研究了铂化合物与铜伴侣蛋白Coxl7,以及亚砷酸钠与多种锌指蛋白的相互作用机制,并且研究了一些重要的细胞环境因素对这些相互作用的影响。第一章是对铂类药物和亚砷化合物的生理学作用机制进行综述。主要涉及铂类药物与DNA靶点的作用以及诱导细胞凋亡的机制；铂类药物的多靶点；铂类药物与铜转运蛋白的关系；亚砷的致癌性和用于癌症治疗的双重功能,以及锌指蛋白与这两种机制的关联。第二章研究了顺铂和人铜伴侣蛋白Cox17的体外和体内的相互作用。我们利用大肠杆菌体系进行了Cox17蛋白的表达,利用Ni-NTA柱和凝胶过滤色谱进行了蛋白的纯化。用紫外-可见光谱、高效液相色谱、电感耦合等离子体质谱和电喷雾质谱的方法研究了Cox172s-s和顺铂的相互作用,同时实验中使用了氧化态的Cox173s-s作为对照,以研究铜结合位点在Cox17与顺铂结合中的功能。结果证实,Cox17蛋白的铜结合位点,也是顺铂的主要结合位点,顺铂通过与铜位点的自由半胱氨酸残基配位形成Pt-S键结合到Cox17上。而且Pt-S键的形成活化了顺铂的其它配位位点,使得顺铂的原始配体很容易离去,而形成四个原始配体都被取代的裸铂加合物{Pt}-COx17。此外,细胞实验证明,Cox17作为一个铜伴侣蛋白,参与顺铂向线粒体的运输。此结果是对铜转运蛋白参与顺铂细胞传递过程的结论的进一步证实和补充。第三章研究了三种典型铂化合物顺铂、反铂、奥沙利铂与Cox172s-s的作用机制,以及关键的生物分子谷胱甘肽(GSH)对三种不同铂化合物与Cox172s-s作用机制的影响。结果显示,三种不同的铂化合物都能够和Cox172s-s铜位点的自由半胱氨酸结合。反铂和奥沙利铂与Cox17的结合方式与顺铂不同,反铂脱去两个原始氯离子而与Cox17形成单一的单铂加合物{Pt(NH3)2}-COx17和双铂加合物{Pt2(NH3)4}-Cox17,奥沙利铂脱去原始草酸根而与Cox17形成单一的单铂加合物{Pt(DACH)}-Cox17和双铂加合物{Pt2(DACH)2}-Cox17。此外,三种铂与含铜蛋白CuICox172s-s的反应,都会引起铜从Cox17上的快速释放。可以推断,在细胞内与铂化合物的结合可能会抑制Cox17的生物学功能。同时我们发现,GSH对三种铂化合物与Cox172s-s的作用具有不同的影响。GSH的存在,对于顺铂、反铂、奥沙利铂和Cox172s-s的结合分别表现出促进、抑制、无影响三种不同的作用。这一现象与顺铂、奥沙利铂和反铂的抗肿瘤活性表现一致。更为重要的是,与GSH的反应之后,反铂和奥沙利铂被失活,顺铂不仅没有被失活,而且表现出更高的与Cox172s-s的反应活性。这些证据证明,GSH对铂类药物的生物学机制的影响可能是十分复杂多样的。第四章研究了亚砷酸钠与C2H2, C3H和C4型锌指的单结构域和多结构域蛋白质的作用,通过荧光方法检测了锌指蛋白与亚砷酸钠反应的亲和力和反应速率、电喷雾质谱检测了反应产物、圆二色光谱和核磁共振检测了亚砷酸钠对锌指结构变化的影响。得到了As(Ⅲ)与锌指蛋白结合的表观解离常数和准一级速率常数、结合产物组成以及、结构变化。结果表明,单锌指结构域蛋白与As(Ⅲ)的反应,在亲和力以及反应速率上都符合C4>C3H>>C2H2的趋势。C3H和C4型锌指比C2H2型对As(Ⅲ)的亲和力高两个数量级,显示出亚砷对锌指类型的选择性。对于多结构域锌指蛋白,多结构域的存在大大提高了C2H2型锌指蛋白对亚砷的亲和力,而对C3H和C4型影响很小,最终三种类型的多结构域蛋白表现出相似的亚砷反应活性。这表明配位位点的数目是影响蛋白与亚砷的亲和性强弱的最为关键的因素。第五章以NCp7-zf2作为模型蛋白研究了As(Ⅲ)、Zn2+对锌指蛋白的竞争关系,以及溶液环境对亚砷对锌指结构的破坏的影响;以Sp1-zf2作为模型蛋白,研究了含巯基生物小分子(GSH、Cys)对亚砷与双配位位点蛋白相互作用的影响。研究表明,亚砷和锌指蛋白的相互作用涉及到As(Ⅲ)、Zn2+对锌指蛋白的竞争结合,中性缓冲液中,Zn2+对锌指蛋白的亲和力远高于As(Ⅲ),所以亚砷不能破坏蛋白的锌指结构。但是,当受到细胞内因素(如pH值降低、GSH、 Cys、His等)的影响时,As(Ⅲ)、Zn2+的竞争会发生改变,As(Ⅲ)有可能取代锌指结构中的Zn2+。细胞因素的这种影响,主要是通过改变Zn2+与锌指蛋白的亲和力实现的,而对As(Ⅲ)与锌指蛋白的亲和力的影响较小。双巯基位点蛋白也是亚砷的重要靶点,在以Sp1-zf2蛋白为模型的研究上发现：GSH、Cys等含巯基分子可能对亚砷与双巯基配位位点蛋白的结合有重要作用。GSH、Cys的存在不仅提高了亚砷与Sp1-zf2结合的亲和力,也加快了二者的反应速率。质谱结果表明,GSH、Cys通过配位到As(Ⅲ)的第三个位点,促进了亚砷与双位点蛋白结合的稳定性。这些结果表明,砷的生理学作用机制可能受到细胞环境的重大影响。更多还原

【Abstract】 Platinum-and arsenic-based drugs are successfully used in clinic for the cancer chemotherapies. The mechanisms of platinum and arsenite drugs are closely related to their interactions with the sulfur-containing of proteins. The sulfur-containing copper transport proteins are proposed to be important for the cellular uptake, transfer, and efflux of platinum drugs. Zinc finger proteins (ZFPs) are proved to be one of the important and direct targets of arsenite compounds. In this dissertation, the interactions of platinum compounds with the copper chaperone Cox17are investigated, and the interaction of arsenite with zinc finger proteins are studied. The influences of celluar small molecules on these interactions were also studied.In the first chapter, the mechanism of platinum drugs and arsenic compounds are reviewed, including the DNA targets of platinum drugs and mechanisms of DNA damage induced apoptosis; the multi-targets of platinum drugs; the relationship of platinum drugs and the copper transport proteins. The dual function of arsenic in both the carcinogenicity and the cancer chemotherapy are discussed.In chapter2, the interaction of cisplatin and human copper chaperone Cox17was investigated. The protein was overexpressed in E. coli and purified using a Ni-NTA column and gel filtration chromatography. UV-visible spectroscopy, High Performance Liquid Chromatography (HPLC), ICP-MS and ESI-MS method were used to study the interaction of Cox172s-s with cisplatin. In order to verify the coordination of copper binding site in the cisplatin reactions, experiments are also performed on the oxidized state Cox173s.s as the control. Results show the copper binding site of Cox17is also the major binding site of cisplatin. The formation of Pt-S bond causes the release of ammine ligands of cisplatin and the nacked platinum binding is observed with long time reaction. In addition, cellular uptake assay on cancer cells demonstrates that copper chaperone protein Cox17is involved in the transport of cisplatin to mitochondrial. This result further confirmed and supplemented the conclusions that copper transport proteins are involved in cell transfer process of cisplatin.In chapter3, the reactions of Cox172s-s with three platinum compounds cisplatin, transplatin, and oxaliplatin have been studied, and the influences of glutathione (GSH) on these reactions have been investigated. Results show that three platinum complexes are able to bind to Cox172s-s at the copper binding site. Transplatin and oxaliplatin bind to Cox17with mode different from cisplatin. Transplatin generates {Pt(NH3)2}-Cox17and {Pt2(NH3)4}-Coxl7adducts, and oxaliplatin generates {Pt(DACH)}-Coxl7and {Pt2(DACH)2} adducts. In addition, three platinum complexes can also react with CuICox172s-s, leading to the rapid release of copper from Cox17. It can be inferred that the binding of platinum complexes can leads to disruption of the function of Cox17. Glutathione shows different effects on the platination of Cox17by different platinum complexes. When the reactions were carried out in the presence of physiological concentration of glutathione, the platination of Cox17by cisplatin, transplatin and oxaliplatin were increased, decreased and unchanged respectively. Moreover, the cisplatin-GSH adducts still show high reactivity to Cox17while transplatin-GSH and oxaliplatin-GSH adducts almost can not react with Cox17. These findings suggest that the interaction of platinum drugs with cellular proteins will be highly affected by glutathione. Moreover, the influences of glutathione are very different depending on platinum species.In chapter4, the reactions of sodium arsenite with different ZFPs have been investigated. The single-domain and multi-domain zinc finger proteins of C2H2, C3H and C4types are expressed for the arsenite reactions. The binding affinity and reaction rate are determined using fluorescence specctroscopy. The product identities are characterized using ESI-MS, and the protein structure perturbations are verified using Circular dichroism and2D1H-15N HSQC NMR spectroscopy. The apparent dissociation constant and the pseudo first order rate constant of the reaction of arsenite and ZFPs are calculated. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4> C3H>> C2H2. Compared with the C2H2motif ZFPs, the binding affinities of C3H and C4motif ZFPs are nearly two orders of magnitude higher and the reaction rates are about two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2type of ZFPs, but has negligible effects on C3H and C4ZFPs. Consequently, the reactivities of three types of multi-domain ZFPs are rather similar. This result indicates that the number of coordination sites of protein is the most critical factor determining the binding affinity.In chapter5, the NCp7-zf2is used as a model protein to investigate the competition of arsenic and zinc binding to ZFPs and the influence of solution conditions on the reaction. Spl-zf2was used as a model protein to investigate the influence of small thiol-containing biomolecules (GSH, Cys) on the interaction of arsenic with two-site protein. Results show that the interaction of arsenic with ZFPs is involved in the competitive binding of arsenic and zinc to ZFPs. Zinc competes overwhelmingly with arsenic for ZFPs in nomal neutral solution, and arsenite can not interferes with zinc finger structure. However, the reaction conditions (such as pH decreases, GSH, Cys, His, etc.) can change the competitive balance of arsenite and Zn2+. Under these conditions, arsenic could replace zinc on ZFPs. The affinity of zinc and ZFPs is greatly affected while the affinity of arsenic and ZFPs is less affected under these conditions. The presence of GSH or Cys not only improves the affinity but also promotes the reaction rate of arsenic binding to Spl-zf2. The mass spectrometry results suggest that GSH or Cys promotes the stability of adducts of arsenite by coodinating to the third site of arsenic. These results suggest that the biological mechanisms of arsenic largely depend on the intracellular environment.更多还原