In vitro folding and assembly of class I MHC molecules

描述（由申请人提供）：近年来，基于细胞的研究在了解内质网（ER）的关键蛋白如何合作折叠和组装I类MHC分子方面非常有用。然而，由于这些研究是从不同的方向进行的，因此不可避免地导致了对第一类成熟的碎片化看法。目前，利用分子和结构方法研究I类组装途径不仅是可能的，而且是统一和推进我们对I类成熟认识的必要条件。该应用程序的长期目标是提供对I类抗原呈递途径的功能，分子和结构方面的综合理解。该项目旨在确定I类抗原呈递途径的机制基础，重点关注tapasin （TPN）的作用。具体目标是：(1)重建I类组装复合物内的相互作用，以深入了解I类分子对肽的选择过程。为此，我们设计了一个可溶的TPN/HLA-B*0801复合物，连接在Jun/Fos链上。TPNjun/HLA- B*0801fos复合物是稳定的，使我们能够首次进行直接肽动力学关联/解离和肽交换实验。将CRT和ERp57或其组装中间体包含到TPNjun/HLA-B*0801fos复合体中，将允许检查这些蛋白如何影响tpn介导的事件；(2)了解I类成熟依赖TPN等位基因差异的分子基础；(3)确定TPN与HLA-B*0801分子的相互作用位点；(4)用原子细节表征CRT中凝集素和多肽结合位点。这些问题将使用诸如基于动力学的分析、定点诱变、圆二色性、荧光、分析性超离心和x射线晶体学等技术来解决。一类mhc限制性免疫反应对控制病毒传播和恶性肿瘤至关重要。因此，了解细胞内的事件如何调节I类分子呈现的肽库是很重要的，因为这些最终影响个体应对挑战的能力。鉴于许多病毒已经进化出干扰内质网I类成熟的策略，了解I类组装途径的进展也将推动病毒免疫逃避机制领域向前发展。我们的研究还将提供关于内质网驻留伴侣在预防蛋白质错误折叠中的作用模式的新知识，这一现象与越来越多的疾病有关。

DESCRIPTION (provided by applicant): In recent years, cell-based studies have been extremely useful in providing an understanding of how key proteins of the endoplasmic reticulum (ER) cooperate together to fold and assemble class I MHC molecules. However, because these studies were carried out from different directions, it inevitably lead to a fragmented view of class I maturation. At present, the use of molecular and structural approaches to study the class I assembly pathway is not only possible but is necessary to unify and advance our knowledge of class I maturation. The long-term goal of this application is to provide an integrated understanding of functional, molecular, and structural aspects of the class I antigen presentation pathway. This project seeks to determine the mechanistic basis of the class I antigen presentation pathway with a focus on the role of tapasin (TPN). The Specific Aims are: (1) to reconstitute interaction within the class I assembly complex in order to gain insights into the process of peptide selection by class I molecules. For this, we designed a soluble TPN/HLA-B*0801 complex tethered to a Jun/Fos linkage. The TPNjun/HLA- B*0801fos complex is stable and enables us to carry out for the first time direct peptide kinetics association/dissociation and peptide exchange experiments. The inclusion of CRT and ERp57, or their assembly intermediaries, to the TPNjun/HLA-B*0801fos complex will permit to examine how these proteins influence TPN-mediated events; (2) to understand the molecular basis of allelic differences in TPN dependencies for class I maturation; (3) to identify interaction sites between TPN and HLA-B*0801 molecules; and (4) to characterize the lectin- and polypeptide-binding sites in CRT with atomic details. These questions will be addressed using techniques such as kinetics-based assays, site-directed mutagenesis, circular dichroism, fluorescence, analytical ultracentrifugation, and x-ray crystallography. Class I MHC-restricted immune responses are essential for controlling the spread of viruses and the malignancy of carcinomas. It is therefore important to understand how events from within cells modulate the peptide repertoire presented by class I molecules since these ultimately impact on the ability of individuals to respond to challenges. Advances in understanding the class I assembly pathway will also move the field of viral immune evasion mechanisms forward given that numerous viruses have evolved strategies to interfere with class I maturation in the ER. Our studies will also provide new knowledge on the mode of action of ER- resident chaperones in preventing protein misfolding, a phenomenon linked to a growing number of diseases.