IN VITRO FOLDING AND ASSEMBLY OF CLASS I MHC MOLECULES

肽填充的I类主要组织相容性（MHC）分子的折叠和组装发生在内质网（ER），是一个多步骤的过程，在结合抗原肽之前，由重链（HC）和β 2微球蛋白（β 2）亚基与伴侣钙连联蛋白和钙连联蛋白、抗原加工转运蛋白（TAP）和蛋白tapasin结合介导。尽管已经确定了折叠途径中涉及的关键蛋白质，但尚不清楚它们是如何相互作用的，以及它们完成了哪些特定的功能。这一建议试图理解与沿着这一途径形成特定中介有关的许多关键方面；HC/ β 2 /钙网蛋白复合物。我们将使用生物化学和晶体学方法的结合：证明可溶性HC/ β 2异二聚体和可溶性伴侣钙网蛋白之间的直接结合；确定控制这种关联的参数；评估伴侣相关HC/beta2m异源二聚体的稳定性和蛋白酶敏感性；探索互动的场所；阐明钙调蛋白的作用和作用方式，因为它与I类MHC分子有关；求解HC/ β 2 /钙网蛋白络合物的晶体结构；在被肽配体占据之前，提供肽结合位点的详细结构描述；并评估结果的含义(1)在控制肽填充的I类MHC分子组装的整体机制的背景下，以及(2)与已知依赖钙网蛋白正确折叠的其他内质网蛋白的关系。这项研究的长期目标是提供对细胞过程的分子理解，这一过程对于维持对病毒和病原体的有效免疫反应至关重要。这些进化的原理对理解胸腺中T细胞的发育、肿瘤免疫学和自身免疫具有重要意义。

Folding and assembly of peptide-filled class I major histocompatibility (MHC) molecules occurs in the endoplasmic reticulum (ER) and is a multi-step process mediated by association of the heavy chain (HC) and beta2microglobulin (beta2m) subunits with the chaperones calnexin and calreticulin, the transporter for antigen processing (TAP), and the protein tapasin, prior to binding antigenic peptides. Although the key proteins involved along the folding pathway have been identified, it is unclear just how they interact with each other and which particular functions they fulfill. This proposal seeks to understand many critical aspects related to the formation of a specific intermediary along this pathway; the HC/beta2m/calreticulin complex. We will use a combination of biochemical and crystallographic approaches to: demonstrate the direct binding between the soluble HC/beta2m heterodimer and the soluble chaperone calreticulin; determine the parameters governing this association; assess the stability and protease sensitivity of the chaperone-associated HC/beta2m heterodimer; probe the sites of interaction; clarify the role and mode of action of calreticulin as it pertains to its association with class I MHC molecules; solve the crystal structure of the HC/beta2m/calreticulin complex; provide a detailed structural description of the peptide binding site prior to being occupied with a peptide ligand; and assess the implications of the results (1) in the context of the overall mechanism governing the assembly of peptide-filled class I MHC molecules, and (2) in relation to other ER proteins known to depend on calreticulin to properly fold. The long term objective of this study is to provide a molecular understanding of a cellular process critical for maintaining effective immune responses against viruses and pathogens. The principles which evolve have implications to understand development of T cells in the thymus, tumor immunology, and autoimmunity.