Towards Structural Studies of Aminopeptidases in Antigen Processing

描述（由申请人提供）：抗原处理是细胞介导的免疫监视和应答的一个组成部分，涉及MHC限制和T细胞识别。经过加工的抗原肽，当MHC分子在细胞表面呈递给T细胞时，作为身份标签被我们的免疫系统监测。因此，抗原的加工和呈递影响个体对感染性有机体的反应，并与疾病的易感性和自身免疫的发展有关。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细胞毒性反应，导致目标细胞的裂解。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加工而成的肽，并将这些肽呈递给细胞毒性T淋巴细胞（ctl），后者可以消除感染细胞，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对抗各种病原体至关重要。为了使表位被T细胞识别，外源抗原需要加工成适当大小的短肽，以便与MHC分子形成物理复合物。第一类抗原肽的前体主要由细胞质中的蛋白酶体产生，然后通过与抗原加工相关的转运体（TAP）转运到内质网（ER）的管腔。最近，内质网内一种名为ERAAP的氨基肽酶被发现是修饰肽前体并产生i类限制性表位的最终n端的缺失环节。我们最近成功地表达和纯化了活性ERAAP酶，并在初步结晶筛选中获得了晶体，这为提供这种关键酶的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，我们提出纯化ERAAP及其子结构域的晶体质量和数量，用于生化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：本提案中获得的生化信息和衍射质量晶体将使ERAAP的高分辨率结构分析成为可能，从而增强我们对抗原加工机制的理解。这种结构信息对于制定生物防御保护战略是必要的，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.