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.

强生公司 (J&J) 投资于五个治疗领域，这些领域对新药开发的需求仍然大量未得到满足。其中之一是抗病毒疗法，现在人们认识到迫切需要设计抑制病毒感染早期阶段的药物。这与当前一代的蛋白酶和聚合酶抑制剂形成鲜明对比，后者在临床上快速选择病毒变体。事实上，这些变异在治疗对象中出现的速度之快表明丙型肝炎病毒（HCV）等病毒正在感染新细胞。拟议的合作解决了该公司开发针对丙肝病毒感染等传染病的新药的战略利益。通过了解关键受体分子的结构和细胞生物学，该项目将出现新的发现管道。我们提出，两种具有 4 个跨膜结构域的膜蛋白 CD81 和 Claudin-1 (CLDN1) 的结构和功能特征将能够阐明它们的蛋白质-蛋白质相互作用的机制。 CLDN1 和 CD81 形成受体，使 HCV 能够感染细胞。这两种共受体成分的寡聚状态对此过程至关重要。我们已经证明 CD81 在质膜上寡聚化，并鉴定出 CD81-CD81 同二聚体和 CD81-CLDN1 异二聚体，表明 CLDN1 被招募到富含 CD81 的结构域。 CD81-CLDN1 复合物的扰动会抑制 HCV 进入，这表明在病毒进入过程中发挥着关键作用。此外，我们最近还鉴定了 CLDN1 第一个细胞外环 (EC1) 中的氨基酸残基，这些残基定义了与 CD81 和病毒辅助受体活性的关联。比尔实验室通过提供合适的重组膜蛋白靶标和不同寡聚状态的突变体来做出贡献。 McKeating 实验室提供这些蛋白质的生物测定方面的专业知识，并可以获得一系列独特的抗体试剂。比尔和麦基廷此前曾共同发表过文章，并正在准备一些额外的手稿。了解这些分子将有助于设计针对病毒感染的治疗剂。我们的具体目标将解决以下问题： A. CD81 的寡聚状态是什么？迄今为止，由于缺乏适当的工具，CD81 及其家族成员（四跨膜蛋白）的寡聚状态很大程度上未知，蛋白质构象与生物活性之间的关系也不清楚。我们假设 HCV 使用二聚体 CD81 进入细胞。我们将测试这一假设并确定定义四跨膜蛋白寡聚化和受体活性的蛋白质基序。我们正在进行的单体和寡聚 CD81 结晶试验（我们有单体 CD81 的衍射晶体）将补充这项工作，因为没有任何一个家族的任何全长成员的结构信息。 B. CLDN1 的寡聚状态如何？我们最近报道了基于标记分子之间的荧光共振能量转移 (FRET) 的 CLDN1 二聚化，表明二聚体是紧密连接链的主要构建模块。生物物理和结构表征将鉴定 HCV 使用的寡聚体状态和构象。 C. CD81-CLDN1 辅助受体复合物的结构组织是什么？我们最近证明酵母表达的 CD81 和 CLDN1 可以结合 HCV。酵母没有 CD81 或 CLDN1 同源物，这使我们能够研究（而不是在哺乳动物细胞中间接）CD81 和 CLDN1 关联以及病毒共受体活性的结构基础。这个膜蛋白生物化学和细胞生物学合作研究项目在药物发现管道的背景下，为所有合作伙伴，尤其是博士生提供了一个千载难逢的机会。