Towards Structural Studies of Aminopeptidases in Antigen Processing

描述（由申请方提供）：抗原加工是涉及MHC限制和T细胞识别的细胞介导的免疫监视和应答的组成部分。加工的抗原肽，当通过MHC分子呈递到T细胞的细胞表面时，作为身份标签被我们的免疫系统监测。因此，抗原加工和呈递影响个体对感染性生物体的反应，并且已经涉及对疾病的易感性和自身免疫的发展。免疫系统消除病毒感染或恶性细胞的一种有效方法是产生导致靶细胞溶解的细胞毒性反应。这种抗病毒机制通常涉及MHC I类分子结合由感染细胞内合成的病毒蛋白质加工的肽，并将这些肽呈递给细胞毒性T淋巴细胞（CTL），这可以消除感染的细胞，从而消除新病毒产生的潜在来源。因此，了解抗原加工的机制对于对抗各种病原体至关重要。对于要被T细胞识别的表位，需要将外来抗原加工成具有适当大小的短肽，以便与MHC分子形成物理复合物。I类抗原肽的前体主要由胞浆中的蛋白酶体产生，然后通过与抗原加工相关的转运蛋白（TAP）转运到内质网（ER）的内腔中。最近，ER内的氨肽酶，命名为ERAAP，已被确定为一个缺失的环节，修剪肽前体和产生的I类限制性表位的最终N-末端。我们最近成功表达和纯化活性ERAAP酶，并在初步结晶筛选中获得晶体，为这种关键酶提供高分辨率结构信息带来了令人兴奋的可能性。在这个探索性（R21）项目中，我们建议纯化ERAAP，其子域和复合物的晶体质量和数量，用于生物化学表征和生长衍射质量晶体，长期目标是获得ERAAP的高分辨率结构。公共卫生相关性：生化信息和衍射质量晶体中获得的建议将使ERAAP的高分辨率结构分析，以提高我们的抗原加工机制的理解。这种结构信息对于制定生物防御的保护战略是必要的，因此对公共卫生至关重要。

DESCRIPTION (provided by applicant): Antigen processing is an integral part of cell-mediated immune surveillance and responses that involve MHC- restriction and T cell recognition. Processed antigenic peptides, when presented on cell surface by MHC molecules to T cells, serve as identity tags to be monitored by our immune system. Thus antigen processing and presentation influence the response of an individual to infectious organisms and has been implicated in the susceptibility to diseases and to the development of autoimmunity. One effective way the immune system eliminates virus-infected or malignant cells is to mount a cytotoxic reaction that result in lysis of target cells. This antiviral mechanism generally involves MHC class I molecules to bind peptides processed from viral proteins synthesized within infected cells, and present those peptides to cytotoxic T lymphocytes (CTLs), which can eliminate the infected cells and thus eliminate potential sources of new viral production. Therefore, understanding the mechanism of antigen processing is critical to fight against various pathogens. For an epitope to be recognized by T cells, foreign antigens need to be processed into short peptides with proper sizes in order to form physical complexes with MHC molecules. The precursors of class I antigenic peptides are generated mainly by proteasomes in the cytosol, and are then transported into the lumen of the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Recently, an aminopeptidase inside the ER, named ERAAP, has been identified to be one missing link that trims peptide precursors and generates the final N-termini of class I-restricted epitopes. Our recent success in expressing and purifying active ERAAP enzyme, and obtaining crystals in the preliminary crystallization screens, have brought about the exciting possibility of providing high-resolution structural information for this critical enzyme. In this exploratory (R21) project, we propose to purify crystallographic quality and quantity of ERAAP, its subdomains, and complexes for biochemical characterization and growing diffraction-quality crystals, with the long-term goal of obtaining high-resolution structures of ERAAP. PUBLIC HEALTH RELEVANCE: Biochemical information and diffraction-quality crystals obtained in this proposal will enable high-resolution structural analyses of ERAAP to enhance our understanding of the mechanisms of antigen processing. This structural information is necessary for the development of protective strategies for biodefense and is thus critical to public health.