【作者】 王玉；

【导师】 刘扬中；

【作者基本信息】 中国科学技术大学 ， 化学生物学， 2023， 博士

【摘要】 蛋白质和核酸是复杂生命过程的重要参与者,与细胞生长、发育以及疾病的发生发展密切相关。本论文从分子层面分别探究了肿瘤相关蛋白质、核酸与金属离子、金属药物之间的相互作用,内容主要涉及三个方面:(1)Cu+与环指蛋白RNF11的相互作用;(2)RNF11影响顺铂毒性的机制性探究;(3)G-四链体、HMGB1以及顺铂与DNA的相分离。相关探究有助于理解蛋白质功能的发挥与疾病的发生发展之间的关系,进而为药物研发、疾病治疗以及解决药物耐药性提供新的思路。第一章是对顺铂的抗肿瘤作用机理、相分离与疾病的分子机制的综述。内容主要包括蛋白质在肿瘤治疗中的潜在药物靶点作用、细胞内铜稳态平衡机制、顺铂与靶点DNA和蛋白质的相互作用以及相分离与肿瘤相关的分子机制。第二章主要研究Cu+与环指蛋白RNF11的相互作用。肿瘤细胞中Cu+含量过高,具有Cu+反应活性的环指蛋白RNF11可能成为其作用靶点,而使蛋白质结构和功能受到影响。我们通过实验发现Cu+与RNF11具有很高的结合能力,导致Zn2+从蛋白质中释放出来,破坏Zn2+介导的环指结构的折叠与稳定,进而抑制环指结构域介导的RNF11与UBE2N的相互作用,对UBE2N/RNF11调控的K63连接多聚泛素化可能产生影响。此外,细胞水平的实验也表明细胞内Cu+水平升高影响RNF11介导的转录调控功能。第三章是对环指蛋白RNF11影响顺铂毒性的机制探究。顺铂是一种多靶点抗癌药物,可以与多种金属蛋白结合,且表现出不同的反应活性。RNF11含有Zn2+结合基序,富含半胱氨酸和组氨酸残基,在肿瘤细胞中过表达。细胞毒性实验发现RNF11过表达降低了顺铂的细胞毒性;因此,我们通过光谱、质谱、核磁波谱等方法探究了顺铂与RNF11之间的相互作用,发现顺铂对RNF11具有很高的反应活性,其能与RNF11的半胱氨酸残基结合,形成不同的铂化产物;顺铂结合导致Zn2+从蛋白质中释放,破坏蛋白质的结构并诱导蛋白质寡聚,进而抑制RNF11与UBE2N的相互作用,这可能对UBE2N/RNF11复合物参与调控的泛素化产生影响。此外,研究还发现Cu+结合促进RNF11与顺铂的相互作用,表明肿瘤细胞中的高含量Cu+可能会使RNF11对顺铂的反应性增加。第四章是对顺铂、HMGB1、G-四链体在DNA形成相分离中的相关探究。研究表明DNA结构变化会影响DNA相分离,而顺铂也可以结合DNA使其结构发生变化,且细胞内DNA损伤病灶处存在相分离现象,因此,我们对顺铂是否会诱导DNA发生相分离进行了基础性探究。实验结果表明,顺铂可以诱导促进DNA发生相分离,随着DNA铂化程度的提高,DNA相分离也得到了极大地促进;顺铂损伤DNA特异性结合蛋白质HMGB1在铂化DNA的相分离中也发挥着促进作用。HMGB1可以抑制原癌基因KRAS的转录表达,并对其驱动的胰腺肿瘤的发生表现出显著抑制作用,但其中涉及的调控机制还需要进一步的探究。因此,我们首先通过多种实验方法验证了 KRAS G-四链体(GQKRAS)与HMGB1之间的相互作用,HMGB1可以促进并稳定GQKRAS;此外,GQKRAS与HMGB1在拥挤环境中发生共相分离,且KRAS以GQ构象存在于液滴中。实验结果表明GQ结构、GQKRAS/HMGB1复合物的形成以及静电相互作用在复合物的相分离中发挥着重要作用。这项工作表明折叠良好的蛋白质与折叠成GQ结构的核酸之间存在相分离行为,可能对其功能的发挥表现出调控作用。更多还原

【Abstract】 As the main participants in complex life processes,proteins and nucleic acids are intimately associated with cell growth,development,and the occurrence and development of diseases.This thesis explores the interactions of tumor-related proteins,nucleic acids with metal ions,and metal drugs,respectively,at the molecular level.It is focused on the following three aspects:(1)the interaction between Cu+and the RING finger protein RNF11,(2)the mechanistic investigations of RNF11 on cisplatin cytotoxicity,and(3)G-quadruplex,HMGB1,cisplatin in DNA phase separation.Related researches could contribute to the understanding of the relationship between protein function and disease occurrence and development,providing new insights into disease treatment,drug development,and overcoming drug resistance.The first chapter provides an overview of the anticancer mechanism of cisplatin and the molecular mechanisms of phase separation and disease.The content mainly covers the potential roles of proteins as drug targets in cancer treatment,the mechanism of copper homeostasis in cells,the interactions between cisplatin and DNA or proteins,as well as the molecular mechanisms of phase separation and its relevance to cancer.The second chapter is mainly focused on the interaction between Cu+and RNF11.The high level of Cu+in tumor cells may render RNF11,which is of Cu+reactivity,a potential target for its action,ultimately affecting the structure and function of the protein.We find that Cu+is of strong binding ability to RNF11,leading to the release of Zn2+from the protein and disrupting the folding and stability of the RING finger mediated by Zn2+coordination.Thus,the interaction of RNF11 with UBE2N mediated by RING finger domain is also interfered,which may affect the K63-linked polyubiquitination regulated by the complex UBE2N/RNF11.Moreover,it is also indicated that elevated Cu+ levels within cells can affect the transcriptional regulatory function mediated by RNF11.The third chapter explores the mechanism of RNF 11 affecting cisplatin cytotoxicity.Cisplatin is a multi-targeted anticancer drug that can bind to a variety of metal proteins,exhibiting different reaction activities.RNF11 contains a Zn2+ binding motif which is rich in cysteine and histidine residues.It is overexpressed in tumor cells.Cellular viability experiment indicate that the overexpression of RNF 11 attenuate the cytotoxicity of cisplatin.Thus,the interaction between cisplatin and RNF11 is studied using spectroscopy,mass spectrometry,nuclear magnetic resonance,and other methods.It is found that cisplatin has high reactivity with RNF11,binding to the cysteine residues of RNF11 to form different platinated adducts.Zn2+is ejected from the protein once cisplatin bind to RNF11,disrupting the protein structure and inducing protein oligomerization.The formation of complex UBE2N/RNF11 is also affected,which may affect the ubiquitination involved in UBE2N/RNF11 complex.In addition,the study find that the coordination of Cu+with RNF11 promotes the RNF11 platination,indicating that the high level of Cu+in tumor cells might increase the reactivity of cisplatin to RNF11.In the fourth chapter,we explores the roles of cisplatin,HMGB1,and Gquadruplex in DNA phase separation.Previous studies suggested that the alteration of DNA structure could trigger DNA phase separation,and cisplatin could alter the DNA structure after binding to it.Additionally,the presence of phase separation was also found in DNA damage foci inside cells.Therefore,we conduct a fundamental study on whether cisplatin could induce DNA phase separation.The experimental results demonstrate that cisplatin could induce and facilitate DNA phase separation.As the degree of DNA platination is exacerbated,the promotion of DNA phase separation is enhanced.Furthermore,the platinated DNA-specific binding protein,HMGB1,also plays a promotional role in the phase separation.Moreover,HMGB1 could inhibit the transcription and expression of the oncogene KRAS,and exhibit inhibitory effect on the development of pancreatic tumors driven by KRAS.However,the regulatory mechanisms involved require further exploration.Therefore,we verify the interaction between KRAS G-quadruplex(GQKRAS)and HMGB1 through a variety of experimental methods and HMGB1 could promote and stabilize the GQKRAS.What’s more,GQKRAS and HMGB1 undergoes co-phase separation under crowded conditions,in which the GQ conformation is still retained within the droplets.It is indicated that the GQ structure,the formation of the GQKRAS/HMGB1 complex,and electrostatic interactions play important roles in the co-phase separation.This work suggests that phase separations could also occur between well-folded proteins and GQ structure nucleic acids,which might regulate their functions.更多还原