IN VITRO FOLDING AND ASSEMBLY OF CLASS I MHC MOLECULES

I 类 MHC 分子的体外折叠和组装

• 批准号: 6510965
• 负责人: MARLENE BOUVIER
• 金额: $ 21.18万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510965
• 关键词: MHC class I antigen; calnexin; calreticulin; endoplasmic reticulum; major histocompatibility complex; myosins; protein binding; protein folding; protein protein interaction; protein structure

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.

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) 中，是一个多步骤过程，由重链 (HC) 和 β2 微球蛋白 (β2m) 亚基与伴侣钙联蛋白和钙网蛋白、抗原加工转运蛋白 (TAP) 和 蛋白质塔帕辛，在结合抗原肽之前。 尽管折叠途径中涉及的关键蛋白质已被识别，但尚不清楚它们如何相互作用以及它们履行哪些特定功能。 该提案旨在了解与沿此路径形成特定中介机构相关的许多关键方面； HC/β2m/钙网蛋白复合物。 我们将结合使用生化和晶体学方法来： 证明可溶性 HC/β2m 异二聚体和可溶性分子伴侣钙网蛋白之间的直接结合；确定管理该协会的参数；评估分子伴侣相关的 HC/β2m 异二聚体的稳定性和蛋白酶敏感性；探测相互作用位点；阐明钙网蛋白的作用和作用方式，因为它与 I 类 MHC 分子相关；解析HC/β2m/钙网蛋白复合物的晶体结构；提供肽结合位点在被肽配体占据之前的详细结构描述；并评估结果的含义（1）在控制肽填充的 I 类 MHC 分子组装的总体机制的背景下，以及（2）与已知依赖钙网蛋白正确折叠的其他 ER 蛋白相关。 这项研究的长期目标是提供对维持针对病毒和病原体的有效免疫反应至关重要的细胞过程的分子理解。 其演化原理对于理解胸腺中 T 细胞的发育、肿瘤免疫学和自身免疫具有重要意义。