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

项目摘要 B肝炎病毒（HBV）的慢性感染是世界范围内人类癌症的主要原因， 与肝硬化和肝细胞癌（HCC）的发展密切相关。17-kDa HBx蛋白 HBV是一种致瘤剂，单独或共同影响多种细胞过程 与它所靶向的蛋白质结合。虽然HBx的致癌潜力已被证实，但其结构 也不知道它介导肝脏相关疾病的分子机制。的主要障碍 其主要原因是溶解性差、与所表征的蛋白质缺乏显著的同源性以及内在紊乱。我们 研究已经成功地获得了高度可溶形式的HBx，并且首次表明HBx是[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，最终将这些分子和结构知识转化为 HBxs的扩展功能库。我们的中心假设是，[Fe-S]簇是一个共同的特点， 所有基因型的HBx。我们建议，[Fe-S]辅因子赋予结构，否则无序 蛋白质和调节蛋白质的反应性和相互作用（至少）三个不同的途径： 相互作用，通过改变HBx的低聚或构象状态，B）氧化还原机制， i）电子转移过程到靶蛋白的辅因子，或ii）作为对细胞免疫应答的调节过程。 c）Fe-或[Fe-S]-转移机制，通过该机制HBx可以充当支架 用于铁运输以调节铁稳态和下游分子途径。我们的具体目标将测试 这些假设通过：（目的1）建立辅助因子的生物学相关形式，如果观察到 [4Fe]（稳定）和[2Fe]（瞬时）形式是生理相关的。我们将确定簇配体，半胱氨酸 残基可能涉及二硫化物，并检查临床突变和大序列缺失如何影响 辅因子和HBx功能。(Aim 2）确定[Fe-S]团簇的类型、位置和对铁的影响。 蛋白质结构（无序到有序转变）以及簇掺入是否驱动蛋白质折叠。如果 如果成功，这一步骤将为通过溶液NMR方法解决HBx的高度寻求的结构奠定基础， 单独或与细胞结合配偶体一起。(Aim 3）在类型和氧化还原之间建立联系 [Fe-S]辅因子的形式和HBx生物活性。这一创新提案的预期总体影响 它将从根本上推进我们对HBx在分子和结构水平上的理解， 目前失踪。由于HBx是开发抗癌药物的潜在靶点，因此确定HBx的表达水平是非常重要的。 [Fe-S]辅因子的作用和连接的结构/功能关系，将收集其在病毒诱导的 发病机制，并提供新的治疗途径。

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.