Molecular mechanism of antigen editing by Class-I MHC Chaperones

总结 我建议开发并应用创新的结构生物学和蛋白质工程工具 研究抗原编辑和显示对主要组织相容性的分子机制 复合体（人类的 MHC 或 HLA）分子。 MHC 编码最多态性的蛋白质 人类基因组，并且与比任何其他区域更多的疾病相关。揭开谜底 这些蛋白质的功能将帮助我们了解自身免疫性疾病，例如糖尿病、多种 硬化症和关节炎，以及对病毒病原体和肿瘤的免疫反应。特别是， MHC (MHC-I) 编码的 I 类蛋白在提醒其他免疫系统方面发挥着关键作用 肽抗原系统，源自自身蛋白质、细胞内病原体或肿瘤，通过 与细胞毒性 CD8 T 细胞表达的克隆型 T 细胞受体 (TCR) 相互作用。关键是 正确整合的 MHC-I 与结合的肽抗原的组装是分子伴侣， 积极为展示的库选择高亲和力肽。除了基础科学优点外， 正如以下所建议的，以原子细节表征这种机制具有重要的临床意义 由于肽加载过程失调、下调而导致的免疫缺陷 肿瘤中伴侣的功能，以及病毒免疫逃避直接靶向伴侣 策略。 尽管进行了大量的功能和结构研究，但使用传统方法 已被证明对于阐明 MHC-I/伴侣复合物的 3D 结构无效 结合肽。这是由于肽相互作用的高度动态性质 伴随的 MHC-I 沟。因此，抗原编辑所需的关键构象变化 仍不完全表征。为了消除这些瓶颈，我开发了一种新的 将 NMR 和冷冻电镜的互补数据集与计算相结合的方法 建模程序 Rosetta，以获得此类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集成建模方法来表征 分子伴侣与人类 HLA 分子形成复合物，并阐明分子伴侣之间的动态转变 控制抗原编辑的肽构象。我们的结构成果将得到进一步探索 使用蛋白质定向进化，并使用功能在细胞环境中明确解决 实验，然后进行详细的蛋白质组分析。作为一个长期目标，我计划使用一种结构- 引导方法来设计具有定制特性的新颖伴侣功能，用于 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.