In vitro folding and assembly of class I MHC molecules

描述(申请人提供)：近年来，基于细胞的研究对于了解内质网(ER)的关键蛋白质如何协同折叠和组装I类MHC分子非常有用。然而，由于这些研究是从不同的角度进行的，这不可避免地导致了对I类成熟度的支离破碎的看法。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，而且对于统一和提高我们对I类成熟的认识也是必要的。这项应用的长期目标是提供对I类抗原递呈途径的功能、分子和结构方面的综合理解。本项目旨在确定I类抗原提呈途径的机制基础，重点是TpN的作用。具体目标是：(1)重建I类组装复合体内的相互作用，以便深入了解I类分子选择多肽的过程。为此，我们设计了一个连接到Jun/Fos链上的可溶性TPN/HLA-B*0801复合体。TPNjun/HLA-B*0801fos复合物是稳定的，使我们能够首次进行直接的多肽动力学缔合/解离和多肽交换实验。将CRT和ERp57或它们的组装中间体包含到TPNjun/HLA-B*0801fos复合体中，将有助于研究这些蛋白是如何影响TPN介导的事件的；(2)了解TPN对I类成熟依赖的等位基因差异的分子基础；(3)确定TPN和HLA-B*0801分子之间的相互作用部位；以及(4)用原子细节表征CRT中的凝集素和多肽结合部位。这些问题将使用基于动力学的分析、定点突变、圆二向色性、荧光、分析超速离心法和X射线结晶学等技术来解决。I类MHC限制性免疫反应对于控制病毒的传播和癌症的恶性是必不可少的。因此，重要的是要了解来自细胞内的事件如何调节I类分子呈现的多肽谱系，因为这些最终会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中I类成熟的策略，在理解I类组装途径方面的进展也将推动病毒免疫逃避机制的领域向前发展。我们的研究还将提供关于内质网驻留伴侣在防止蛋白质错误折叠方面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.