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.

项目概要 乙型肝炎病毒 (HBV) 的慢性感染是全世界人类癌症的主要原因，并且 17-kDa HBx 蛋白与肝硬化和肝细胞癌 (HCC) 的发展密切相关。 HBV 是一种致病性致瘤剂，可单独或共同影响多个细胞过程 尽管 HBx 的致癌潜力已得到证实，但其结构却未得到证实。 它介导肝脏相关疾病的分子机制也是未知的。 其缺点是溶解度小、与特征蛋白质缺乏显着同源性以及内在无序。 研究已成功获得高度可溶形式的 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。 驱动蛋白质构象和反应性，最终将这种分子和结构知识转化为 HBxs 的扩展功能库是我们的中心假设是 [Fe-S] 簇是 HBxs 的一个共同特征。 我们认为 [Fe-S] 辅因子赋予了无序的结构。 蛋白质并通过（至少）三种不同的途径调节蛋白质反应性和相互作用：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) 目标蛋白辅因子的电子转移过程或 ii) 作为细胞响应的调节过程 氧化还原状态和 ROS 的生成，c) Fe- 或 [Fe-S]- 转移机制，通过该机制 HBx 可以充当支架 我们将测试铁运输以调节铁稳态和下游分子途径。 这些假设通过：（目标 1）建立辅因子的生物学相关形式，并且如果两者都观察到 [4Fe]（稳定）和[2Fe]（瞬时）形式具有生理相关性，我们将识别簇配体，半胱氨酸。 可能涉及二硫键的残基，并检查临床突变和大序列缺失可能如何影响 辅助因子以及 HBx 功能（目标 2）确定 [Fe-S] 簇的类型、位置和影响。 蛋白质结构（无序到有序的转变）以及簇合并是否驱动蛋白质折叠。 成功后，这一步将为通过溶液核磁共振方法解决备受追捧的 HBx 结构奠定基础， 单独或与细胞结合伙伴一起（目标 3）在类型和氧化还原之间建立联系。 [Fe-S] 辅因子和 HBx 生物活性的形式这一创新提案的预期总体影响。 它将从根本上增进我们对 HBx 在分子和结构水平上的理解，即 由于HBx是抗癌药物开发的潜在靶点，因此目前尚不清楚。 [Fe-S] 辅助因子的作用以及相关的结构/功能关系，将收集其在病毒诱导中的作用 发病机制并提供新的治疗途径。