How does a metallocofactor in the Hepatitis B viral protein X orchestrate pathogenesis and liver cancer?

乙型肝炎病毒 X 蛋白中的金属辅因子如何协调发病机制和肝癌？

• 批准号: 10428574
• 负责人: Maria-Eirini Pandelia
• 金额: $ 33.81万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428574
• 关键词: ATP phosphohydrolase; Address; Affect; Antineoplastic Agents; Antiviral Therapy; Apoptosis; Automobile Driving; Binding; Binding Proteins; Biochemical; Biological; Cancer Etiology; Cell Cycle; Cell physiology; Chemicals; Chemistry; Cirrhosis; Clinical; Complex; Cysteine; DNA Repair; Data; Development; Disease; Disulfides; Electron Transport; Etiology; Event; Generations; Genetic Transcription; Genotype; Glean; Goals; Hepatitis; Hepatitis B; Hepatitis B Virus; Homeostasis; Human; In Vitro; Iron; Iron Regulatory Protein 1; Knowledge; Ligands; Ligation; Link; Liver; Location; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Metalloproteins; Methods; Molecular; Molecular Conformation; Molecular Structure; Mutation; Nature; Oncogenic; Oxidation-Reduction; PTEN gene; Pathogenesis; Pathway interactions; Phosphoric Monoester Hydrolases; Physiological; Play; Primary carcinoma of the liver cells; Process; Property; Protein Conformation; Proteins; Reactive Oxygen Species; Reporting; Research; Role; Sequence Deletion; Sequence Homology; Series; Solubility; Structure; Structure-Activity Relationship; TP53 gene; Techniques; Testing; Time; Translating; Tumor Suppressor Proteins; Variant; Viral; Viral Proteins; Virus; biophysical techniques; cellular targeting; chronic infection; cofactor; crosslink; disulfide bond; experimental study; gene product; genetic regulatory protein; helicase; innovation; insight; mortality; novel strategies; novel therapeutics; protein folding; protein function; protein protein interaction; protein structure; reconstitution; response; scaffold; three dimensional structure; trafficking; tumor; tumorigenesis; tumorigenic

Project Summary Chronic infection by the Hepatitis B virus (HBV) is a leading cause of human cancer worldwide, and is strongly associated with development of cirrhosis and hepatocellular carcinoma (HCC). The 17-kDa HBx protein of HBV is a causative tumorigenic agent and affects multiple cellular processes, either on its own or together with the proteins it targets. Though the oncogenic potential of HBx has been demonstrated, neither its structure nor the molecular mechanisms by which it mediates liver-associated diseases are known. The major obstacles have been the sparing solubility, lack of significant homology to characterized proteins and intrinsic disorder. Our studies have succeeded in obtaining HBx in highly soluble forms and for the first time shown that HBx is an [Fe- S]-binding protein. Our long-term goal is to establish the chemical nature of the cofactor and its involvement in driving protein conformation and reactivities, ultimately translating this molecular and structural knowledge to HBxs’ extended functional repertoire. Our central hypothesis is that the [Fe-S] cluster is a common feature of HBxs across all genotypes. We propose that the [Fe-S] cofactor confers structure in an otherwise disordered protein and modulates protein reactivity and interactions by (at least) three distinct pathways: a) protein-protein interactions, by changing the oligomeric or conformational status of HBx, b) redox mechanisms involving either i) electron transfer processes to cofactors of target proteins or ii) regulatory processes as a response to cellular redox status and generation of ROS, c) Fe- or [Fe-S]- transfer mechanisms, by which HBx can act as a scaffold for iron-trafficking to regulate iron homeostasis and downstream molecular pathways. Our specific aims will test these hypotheses by: (Aim 1) establishing the biologically relevant form of the cofactor, and if both observed [4Fe] (stable) and [2Fe] (transient) forms are physiologically relevant. We will identify the cluster ligands, cysteine residues likely involved in disulfides and examine how clinical mutations and large sequence deletions may affect the cofactor and thus HBx function. (Aim 2) Establish the type, location and effects of the [Fe-S] cluster on the protein structure (disorder-to-order transition) and whether cluster incorporation drives protein folding. If successful, this step will set the stage for solving by solution NMR methods the highly sought structure of HBx, either on its own or together with cellular binding partners. (Aim 3) Establish a link between the type and redox form of the [Fe-S] cofactor and HBx biological activity. The expected overall impact of this innovative proposal is that it will fundamentally advance our understanding of HBx on the molecular and structural level, which is currently missing. Because HBx is a potential target for the development of anti-cancer drugs, determining the role(s) of the [Fe-S] cofactor and the linked structure/function relationships, will glean its part in viral-induced pathogenesis and offer new therapeutic avenues.

项目摘要 乙肝病毒的慢性感染是全球人类癌症的主要原因，而且 与肝硬变和肝细胞癌的发展密切相关。17 kDa HBx蛋白 是一种致癌因子，可单独或共同影响多种细胞过程。 它所针对的蛋白质。虽然HBx的致癌潜力已经被证明，但它的结构 也不知道它介导肝脏相关疾病的分子机制。主要障碍是 一直以来，蛋白质的溶解性较差，缺乏显著的同源性和固有的无序性。我们的 研究已经成功地获得了高度可溶的HBx，并首次表明HBx是一种[Fe- S]-结合蛋白。我们的长期目标是确定辅因子的化学性质及其参与 推动蛋白质构象和反应，最终将这种分子和结构知识转化为 HBxs的扩展功能曲目。我们的中心假设是[Fe-S]星系团是 所有的基因类型都有HBx。我们认为[Fe-S]辅因子赋予了一个原本无序的结构 蛋白质通过(至少)三条不同的途径调节蛋白质的反应性和相互作用：a)蛋白质-蛋白质 相互作用，通过改变HBx的寡聚或构象状态，b)氧化还原机制，涉及 I)电子转移到目标蛋白的辅因子的过程或ii)作为对细胞的反应的调节过程 氧化还原状态和ROS的生成，c)Fe-或[Fe-S]-转移机制，通过这些机制，HBx可以作为支架 用于铁的运输，以调节铁的动态平衡和下游分子通路。我们的具体目标将考验 这些假设通过：(目标1)建立生物上相关的辅因子形式，如果两者都观察到的话 [4Fe](稳定)和[2Fe](暂态)形式在生理上是相关的。我们将确定簇状配体半胱氨酸 可能与二硫化物有关的残基，并检查临床突变和大序列缺失可能会如何影响 辅因子，从而发挥HBx功能。(目标2)确定[Fe-S]星团的类型、位置和对 蛋白质结构(无序到有序的转变)以及簇掺入是否驱动蛋白质折叠。如果 如果成功，这一步骤将为用溶液核磁共振方法解决HBX的高度寻找的结构奠定基础， 可以单独或与细胞结合伙伴一起使用。(目标3)在类型和氧化还原之间建立联系 [Fe-S]辅因子的形式与HBx生物活性。这一创新提议的预期总体影响 它将从根本上促进我们在分子和结构层面上对HBX的理解，这是 目前失踪。由于HBX是抗癌药物开发的潜在靶点，因此确定 [Fe-S]辅因子的作用(S)及其连接的结构/功能关系，将收集其在病毒诱导中的作用 并提供了新的治疗途径。