Molecular mechanism of antigen editing by Class-I MHC Chaperones

摘要 我建议开发和应用创新的结构生物学和蛋白质工程工具来 探讨主要组织相容性抗原编辑和展示的分子机制 复杂的(人类的MHC或人类白细胞抗原)分子。MHC编码的多态蛋白是 人类基因组，与其他任何地区相比，与更多的疾病有关。揭开 这些蛋白的功能将有助于我们了解自身免疫性疾病，如糖尿病、多发性 硬化症和关节炎，以及对病毒病原体和发展中的肿瘤的免疫反应。特别是， 由MHC(MHC-I)编码的第I类蛋白在提醒免疫的其余部分中起着关键作用 来自自身蛋白、细胞内病原体或肿瘤的多肽抗原系统，通过 与细胞毒性CD8T细胞表达的克隆型T细胞受体相互作用。解决问题的关键 正确构象的MHC-I与结合的多肽抗原的组装是分子伴侣 主动为显示的曲目选择高亲和力的多肽。除了基本的科学价值外， 从原子细节上描述这种机制具有重要的临床意义，如下所示 由于肽装载过程的失调而导致的免疫缺陷，下调 分子伴侣在肿瘤中的作用，以及病毒免疫逃避对分子伴侣的直接靶向 战略。 尽管进行了大量的功能和结构研究，但使用常规方法 已被证明对阐明MHC-I/伴侣复合体的3D结构以及 结合多肽。这是由于多肽相互作用的高度动态性质 有陪伴的MHC-I沟槽。因此，抗原编辑所需的关键构象变化 仍然是不完全的特征。为了消除这些瓶颈，我开发了一种新的 将来自核磁共振和CryoEM的互补数据集与计算 建模程序Rosetta，以获得这种具有挑战性的复合体的高分辨率结构。 在这里，我建议应用这种强大的综合建模方法来表征 分子伴侣与人类人类白细胞抗原分子的结合，并阐明两者之间的动态转变 支配抗原编辑的多肽构象。我们将进一步探索我们的结构性成果 使用蛋白质指导的进化，并在细胞环境中使用功能 实验，然后进行详细的蛋白质组学分析。作为一个长期目标，我计划使用一种结构- 用于设计具有定制特性的新型伴侣功能的指导性方法 针对移植物排斥、自身免疫性疾病、病原体的新兴免疫治疗应用 感染和癌症。

Summary I propose to develop and apply innovative structural biology and protein engineering tools to investigate the molecular mechanism of antigen editing and display on major histocompatibility complex (MHC or HLA in humans) molecules. The MHC encodes the most polymorphic proteins in the human genome, and is associated with more diseases than any other region. Unravelling the function of these proteins will help us understand autoimmune diseases, such as diabetes, multiple sclerosis and arthritis, and immune responses to viral pathogens and developing tumors. In particular, Class-I proteins encoded by the MHC (MHC-I) play a pivotal role in alerting the rest of the immune system to peptide antigens, derived from self-proteins, intracellular pathogens or tumors, by interacting with clonotypic T cell receptors (TCRs) expressed by cytotoxic CD8+ T cells. Key to the assembly of properly conformed MHC-I with bound peptide antigen are molecular chaperones that actively select high-affinity peptides for the displayed repertoire. Besides the basic science merit, characterizing this mechanism in atomic detail has important clinical implications, as suggested by immunodeficiencies resulting from dysregulation of the peptide-loading process, the downregulation of chaperone functions in tumors, and the direct targeting of chaperones by viral immune evasion strategies. Despite a large number of functional and structural studies, the use of conventional methods has proven ineffective for elucidating the 3D structure of the MHC-I/chaperone complex together with bound peptides. This is due to the highly dynamic nature of peptide interactions within the chaperoned MHC-I groove. As a result, the crucial conformational changes needed for antigen editing remain incompletely characterized. To remove these bottlenecks, I have developed a new methodology that combines complementary datasets from NMR and cryoEM with the computational modeling program, Rosetta, to obtain high-resolution structures of such challenging complexes. Here, I propose to apply this powerful integrative modeling approach to characterize chaperone complexes with human HLA molecules, and to elucidate dynamic transitions between peptide conformations which govern antigen editing. Our structural results will be further explored using protein directed evolution, and explicitly addressed in a cellular context using functional experiments, followed by a detailed proteomic analysis. As a long-term goal, I plan to use a structure- guided approach to engineer novel chaperone functions with custom specificities, to be used in emerging immunotherapy applications against graft rejection, autoimmune diseases, pathogen infection and cancer.