Structural studies of the MHC class I peptide loading complex

描述（由申请人提供）：在针对病毒和肿瘤的免疫应答的核心过程中，MHC I类分子在细胞表面呈递肽抗原，以供CD 8 + T细胞识别。MHC I类分子在内质网（ER）中装载有肽，同时被隔离在肽装载复合物或PLC内。PLC由抗原加工相关转运蛋白（TAP）和ER-腔亚复合物组成，TAP将抗原肽转运到ER中。该管腔亚复合物足以用于肽加载以及用于低亲和力交换为高亲和力肽，或“肽编辑”。除了MHC I类分子外，亚复合物还包含凝集素伴侣钙网蛋白、蛋白质二硫键异构酶ERp 57和Tapasin（一种MHC I类特异性伴侣）。在缺乏整个亚复合物的结构的情况下，管腔PLC亚复合物的组分如何合作以促进肽加载和编辑一直难以理解。在这里，我们建议获得这一关键的结构信息，为整个ER管腔PLC子复合物作为一种手段，以了解其功能的分子机制。第一个目标是获得足够的材料进行结构测定。目前，我们能够从足够用于单粒子电子显微镜（EM）的数量的组分中重建复合物，我们的挑战是扩大晶体学研究。我们还在开发第二种方法，其中管腔亚复合物的组分在昆虫或人类淋巴母细胞中组装，然后分离整个亚复合物。第二个目的是确定ER腔PLC子复合物的结构，无论是通过X射线晶体学或EM。来自EM的低分辨率信息将显示tapasin、ERp 57和钙网蛋白如何围绕MHC I类分子排列，从而提供关于它们在装载过程中的功能的线索。X射线结构将揭示这些蛋白质如何与MHC I类肽结合沟相互作用的细节，从而表明PLC促进肽装载和交换的机制。PLC结构的理解不仅与MHC I类成熟相关，而且更普遍地与理解ER中的蛋白质折叠和质量控制相关，因为ERp 57和钙网蛋白是不限于MHC I类分子的一般折叠酶。 公共卫生相关性：MHC I类分子在细胞表面呈递肽抗原以供免疫系统识别，在对抗病毒和肿瘤的应答中起关键作用。I类分子通过肽加载复合物加载抗原肽。我们建议可视化这个复杂的，以了解肽加载发生的分子机制。

DESCRIPTION (provided by applicant): In a process central to the immune response against viruses and tumors, MHC class I molecules present peptide antigens at the cell surface for recognition by CD8+ T cells. MHC class I molecules are loaded with peptide in the endoplasmic reticulum (ER) while sequestered within the peptide loading complex, or PLC. The PLC consists of the Transporter associated with Antigen Processing (TAP), which translocates antigenic peptides into the ER, and an ER-luminal subcomplex. This luminal subcomplex is sufficient for peptide loading as well as for the exchange of low affinity for high affinity peptides, or "peptide editing". In addition to the MHC class I molecule, the subcomplex comprises the lectin chaperone calreticulin, the protein disulfide isomerase ERp57, and tapasin, an MHC class I specific chaperone. How the components of luminal PLC subcomplex cooperate to facilitate peptide loading and editing has been difficult to understand in the absence of a structure for the whole subcomplex. Here we propose to obtain this critical structural information for the entire ER-luminal PLC subcomplex as a means to understanding the molecular mechanisms underlying its function. The first aim is to obtain sufficient material for structure determination. At present, we are able to reconstitute the complex from components in quantities sufficient for single particle electron microscopy (EM), and our challenge is to scale up for crystallographic studies. We are also developing a second approach, where the components of the luminal subcomplex are assembled in insect or human lymphoblastoid cells and the entire subcomplex is then isolated. The second aim is to determine the structure of the ER-luminal PLC subcomplex, either by X-ray crystallography or by EM. Low resolution information from EM will show how tapasin, ERp57, and calreticulin are arranged around the MHC class I molecule, thereby providing clues as to their function in the loading process. An X-ray structure will reveal details regarding how these proteins interact with the MHC class I peptide binding groove and would thus suggest the mechanism by which the PLC facilitates peptide loading and exchange. An understanding of PLC architecture is relevant not only for MHC class I maturation but also more generally for understanding protein folding and quality control in the ER, since ERp57 and calreticulin are general foldases that are not restricted to the MHC class I molecule. PUBLIC HEALTH RELEVANCE: MHC class I molecules present peptide antigens at the cell surface for recognition by the immune system, playing a critical role in the response against viruses and tumors. The class I molecules are loaded with antigenic peptides by the peptide loading complex. We propose to visualize this complex in order to understand the molecular mechanisms by which peptide loading takes place.