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核心过程中，MHC I 类分子在细胞表面呈递肽抗原以供 CD8 T 细胞识别。 MHC I 类分子在内质网 (ER) 中装载肽，同时隔离在肽装载复合物 (PLC) 内。 PLC 由与抗原加工 (TAP) 相关的转运蛋白 (TAP) 和 ER 腔亚复合体组成，TAP 将抗原肽转运到 ER 中。该腔亚复合物足以进行肽加载以及将低亲和力肽交换为高亲和力肽，或“肽编辑”。除了 MHC I 类分子外，该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 ERp57 和 Tapasin（MHC I 类特异性伴侣）。在缺乏整个子复合物结构的情况下，很难理解腔 PLC 子复合物的各个组件如何协作以促进肽加载和编辑。在这里，我们建议获得整个 ER-luminal PLC 子复合体的关键结构信息，作为理解其功能背后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，我们能够从足以进行单粒子电子显微镜（EM）的数量的成分重建复合物，我们的挑战是扩大晶体学研究的规模。我们还在开发第二种方法，其中腔亚复合物的成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。第二个目标是通过 X 射线晶体学或 EM 确定 ER-luminal PLC 子复合体的结构。来自 EM 的低分辨率信息将显示 tapasin、ERp57 和钙网蛋白如何排列在 MHC I 类分子周围，从而提供有关它们在加载过程中的功能的线索。 X 射线结构将揭示有关这些蛋白质如何与 MHC I 类肽结合槽相互作用的细节，从而表明 PLC 促进肽装载和交换的机制。了解 PLC 架构不仅与 MHC I 类成熟相关，而且更广泛地与了解 ER 中的蛋白质折叠和质量控制相关，因为 ERp57 和钙网蛋白是不限于 MHC I 类分子的通用折叠酶。 公共卫生相关性：MHC I 类分子在细胞表面呈递肽抗原，供免疫系统识别，在对抗病毒和肿瘤的反应中发挥着关键作用。 I类分子通过肽负载复合物负载抗原肽。我们建议将这种复合物可视化，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.