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反应的中心过程中，MHC I类分子将多肽抗原呈递到细胞表面，供CD8 T细胞识别。MHC I类分子在内质网(ER)中负载多肽，而在多肽负载复合体(PLC)中隔离。PLC由与抗原处理相关的转运蛋白(TAP)和内质网亚复合体组成，TAP负责将抗原肽转移到内质网。这种腔内亚复合体足以负载多肽以及将低亲和力的多肽交换为高亲和力的多肽，或称“多肽编辑”。除了MHC I类分子外，该亚复合体还包括凝集素伴侣钙网蛋白、蛋白质二硫键异构酶ERp57和Tapasin，Tapasin是MHC I类特异性伴侣。在缺乏整个亚复合体的结构的情况下，腔PLC亚复合体的组件如何协作以促进肽的装载和编辑一直是很难理解的。在这里，我们建议获得整个内质网-腔PLC亚复合体的关键结构信息，作为了解其功能的分子机制的一种手段。第一个目标是获得足够的材料来确定结构。目前，我们能够从足以用于单粒子电子显微镜(EM)的组分重建络合物，我们的挑战是扩大结晶学研究的规模。我们还在开发第二种方法，在昆虫或人类淋巴母细胞中组装管腔亚复合体的组件，然后分离整个亚复合体。第二个目标是通过X射线结晶学或EM确定内质网-管腔PLC亚复合体的结构。来自EM的低分辨率信息将显示Tapasin、ERp57和Calreticrin如何排列在MHC I类分子周围，从而为它们在负载过程中的功能提供线索。X射线结构将揭示这些蛋白质如何与MHC I类肽结合槽相互作用的细节，从而提示PLC促进肽负载和交换的机制。了解PLC的结构不仅与MHC I类分子的成熟有关，而且更广泛地与理解内质网中的蛋白质折叠和质量控制有关，因为ERp57和钙网蛋白是一般的折叠酶，不限于MHC I类分子。 公共卫生相关性：MHC I类分子在细胞表面呈递多肽抗原，供免疫系统识别，在对抗病毒和肿瘤的反应中发挥关键作用。I类分子通过肽负载复合体负载抗原肽。我们建议将这种复合体可视化，以了解多肽负载发生的分子机制。

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.