Structural characterisation of cd81-claudin1 hepatitis c cirus receptor complex

cd81-claudin1 丙型肝炎病毒受体复合物的结构表征

• 批准号: BB/H016651/1
• 金额: $ 9.59万
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Training Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH016651%2F1
• 关键词: Structural; characterisation; cd81; claudin1; hepatitis

Johnson & Johnson (J&J) invests in five therapeutic areas where there continues to be significant unmet need for novel drug development. One of these is anti-viral therapies, where it is now recognised that there is an urgency to design drugs that inhibit the early steps of viral infection. This contrasts with the current generation of protease and polymerase inhibitors which rapidly select viral variants in the clinic. Indeed, the rapidity at which these variants appear in treated subjects suggests viruses such as hepatitis C virus (HCV) are infecting new cells. The proposed collaboration addresses the company's strategic interest in developing new drugs targeted at infectious diseases such as HCV infections. By gaining an understanding of the structural and cell biology of key receptor molecules, novel discovery pipelines will emerge from this project. We propose that structural and functional characterisation of two membrane proteins each with 4 transmembrane domains, CD81 and Claudin-1 (CLDN1), will enable the mechanism underlying their protein-protein interactions to be elucidated. CLDN1 and CD81 form a receptor that enables HCV to infect cells. The oligomerisation status of these two co-receptor components is critical to this process. We have shown that CD81 oligomerises at the plasma membrane, with the identification of CD81-CD81 homodimers and CD81-CLDN1 heterodimers suggesting a recruitment of CLDN1 to CD81-enriched domains. Perturbation of CD81-CLDN1 complexes inhibits HCV entry, suggesting a critical role in the viral entry process. Furthermore, we have recently identified the amino acid residues in the first extracellular loop (EC1) of CLDN1 that define association with CD81 and viral co-receptor activity. The Bill laboratory contributes by providing access to suitable recombinant membrane protein targets and mutants of varying oligomeric states. The McKeating laboratory provides expertise in biological assays for these proteins, as well as having access to a range of unique antibody reagents. Bill and McKeating have previously published together, and have a number of additional manuscripts in preparation. Understanding these molecules will aid the design of therapeutic agents targeting viral infection. Our specific aims will address the following questions: A. What is the oligomeric status of CD81? To date, because of the lack of appropriate tools, the oligomeric status of CD81 and its family members (the tetraspanins) is largely unknown and the relationship between protein conformation and biological activity is poorly defined. We hypothesise that HCV uses dimeric CD81 to enter cells. We will test this hypothesis and identify protein motifs defining tetraspanin oligomerisation and receptor activity. Our ongoing crystallisation trials of monomeric and oligomeric CD81 (we have diffracting crystals of monomeric CD81) will complement this work, since there is no structural information available for any full-length member of either family. B. What is the oligomeric status of CLDN1? We recently reported CLDN1 dimerisation based on fluorescence resonance energy transfer (FRET) between tagged molecules, suggesting that dimers are the primary building block(s) of tight junction strands. Biophysical and structural characterisation will identify the oligomeric status and conformation used by HCV. C. What is the structural organisation of the CD81-CLDN1 co-receptor complex? We recently demonstrated that yeast-expressed CD81 and CLDN1 can bind HCV. Yeast does not have CD81 or CLDN1 homologues, allowing us to study (rather than indirectly in mammalian cells) the structural basis of CD81 and CLDN1 association and viral co-receptor activity. This collaborative research programme in membrane protein biochemistry and cell biology, in the context of the drug discovery pipeline, provides an exiting opportunity for all partners and especially a PhD student.

Johnson＆Johnson（J＆J）投资于五个治疗领域，在那里继续对新型药物开发的需求仍然很大。其中一种是抗病毒疗法，现在已经认识到，有迫切需要抑制病毒感染的早期步骤的药物。这与当前一代的蛋白酶和聚合酶抑制剂相反，这些蛋白酶和聚合酶抑制剂在临床中迅速选择病毒变异。实际上，这些变体在治疗受试者中出现的速度表明诸如丙型肝炎病毒（HCV）之类的病毒正在感染新细胞。拟议的合作旨在解决该公司在开发针对传染病（例如HCV感染）的新药方面的战略兴趣。通过了解关键受体分子的结构和细胞生物学，将从该项目中出现新的发现管道。我们提出，两种膜蛋白的结构和功能表征每个具有4个跨膜结构域CD81和Claudin-1（Cldn1），将使其蛋白质相互作用的基础机制阐明。 CLDN1和CD81形成一种受体，使HCV能够感染细胞。这两个共受体成分的寡聚状态对此过程至关重要。我们已经表明，CD81在质膜处的寡聚，并鉴定了CD81-CD81同型二聚体和CD81-CLDN1异二聚体，这表明将CLDN1募集到CD81-富集域。 CD81-CLDN1复合物的扰动抑制了HCV的进入，这表明在病毒进入过程中起关键作用。此外，我们最近在CLDN1的第一个细胞外环（EC1）中确定了与CD81和病毒共受体活性相关的氨基酸残基。法案实验室通过提供对不同寡聚状态的合适重组膜蛋白靶标和突变体的访问来做出贡献。 McKeating实验室为这些蛋白质的生物测定法提供了专业知识，并可以使用一系列独特的抗体试剂。 Bill和McKeating之前已经一起出版了，并准备了许多其他手稿。了解这些分子将有助于设计靶向病毒感染的治疗剂。我们的具体目的将解决以下问题：A。CD81的寡聚状态是什么？迄今为止，由于缺乏适当的工具，CD81及其家庭成员（四翼生蛋白酶）的寡聚状态在很大程度上是未知的，并且蛋白质构象与生物学活性之间的关系很差。我们假设HCV使用二聚体CD81进入细胞。我们将检验该假设，并确定定义四叠蛋白酶寡聚和受体活性的蛋白质基序。我们正在进行的单体和低聚CD81（单体CD81的衍射晶体）的结晶试验将补充这项工作，因为任何一个家庭的全长构件都没有可用的结构信息。 B. CLDN1的低聚状态是什么？我们最近报告了基于标记分子之间的荧光共振能传递（FRET）的CLDN1二聚化，这表明二聚体是紧密连接链的主要构建块。生物物理和结构表征将确定HCV使用的寡聚状态和构象。 C. CD81-CLDN1共受体复合物的结构组织是什么？我们最近证明了酵母表达的CD81和CLDN1可以结合HCV。酵母没有CD81或CLDN1同源物，使我们能够研究（而不是间接地在哺乳动物细胞中）CD81和CLDN1关联以及病毒性共受体活性的结构基础。在药物发现管道的背景下，该膜蛋白生物化学和细胞生物学方面的合作研究计划为所有伴侣，尤其是博士生提供了一个退出的机会。