Molecular mechanism of antigen editing by Class-I MHC Chaperones

总结 我建议开发和应用创新的结构生物学和蛋白质工程工具， 探讨抗原编辑和展示对主要组织相容性影响的分子机制 复合物（人类的MHC或HLA）分子。MHC编码的多态性最高的蛋白质， 人类基因组，与更多的疾病比任何其他地区。解开 这些蛋白质的功能将帮助我们了解自身免疫性疾病，如糖尿病，多 硬化和关节炎，以及对病毒病原体的免疫反应和发展中的肿瘤。特别是， 由MHC编码的I类蛋白（MHC-I）在警告免疫系统的其余部分中起着关键作用。 系统肽抗原，来自自身蛋白质，细胞内病原体或肿瘤， 与细胞毒性CD 8 + T细胞表达的克隆型T细胞受体（TCR）相互作用。的关键 正确整合的MHC-I与结合的肽抗原的组装是分子伴侣， 主动选择高亲和力肽用于展示的库。除了基本的科学价值， 在原子细节上表征这种机制具有重要的临床意义，正如 由于肽装载过程的失调而导致的免疫缺陷， 分子伴侣在肿瘤中的功能，以及通过病毒免疫逃避直接靶向分子伴侣 战略布局 尽管进行了大量的功能和结构研究， 已经证明对于阐明MHC-I/伴侣蛋白复合物的3D结构无效， 结合肽。这是由于肽相互作用的高度动态性质， MHC-I型分子伴侣因此，抗原编辑所需的关键构象变化 仍然不完全特征化。为了消除这些瓶颈，我开发了一种新的 将NMR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物的高分辨率结构。 在这里，我建议应用这种强大的综合建模方法来表征 分子伴侣复合物与人类HLA分子，并阐明动态转换之间 控制抗原编辑的肽构象。我们的结构结果将进一步探讨 使用蛋白质定向进化，并在细胞环境中使用功能性 实验，然后进行详细的蛋白质组学分析。作为一个长期目标，我计划使用一个结构- 指导方法，以定制特异性设计新颖的伴侣蛋白功能，用于 针对移植排斥、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.