Towards Structural Studies of Aminopeptidases in Antigen Processing

抗原加工中氨基肽酶的结构研究

• 批准号: 7877060
• 负责人: Hwai-Chen Guo
• 金额: $ 3.99万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-19 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7877060
• 关键词: Aminopeptidase; Antigen Presentation Pathway; Antigens; Antiviral Agents; Autoimmunity; Baculoviruses; Binding; Biochemical; Cell surface; Cells; Complex; Crystallization; Cytolysis; Cytosol; Cytotoxic T-Lymphocytes; Development; Disease; Endoplasmic Reticulum; Enzymes; Epitopes; Failure; Funding; Goals; Histocompatibility Antigens Class I; Human; Immune system; Immunologic Surveillance; Individual; Infectious Agent; Insecta; Link; Mediating; Monitor; N-terminal; Names; Peptide/MHC Complex; Peptides; Predisposition; Process; Production; Proteins; Proteolysis; Public Health; Reaction; Recombinant Proteins; Resolution; Series; Source; Specificity; Structure; System; T-Lymphocyte; Variant; Viral; Viral Proteins; Virus; analog; antigen processing; antigenic peptide transporter; biodefense; cancer cell; cytotoxic; design and construction; experience; fight against; improved; inhibitor/antagonist; multicatalytic endopeptidase complex; pathogen; public health relevance; puromycin-insensitive leucyl-specific aminopeptidase; response; scale up; success

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.

描述（由申请人提供）：抗原加工是细胞介导的免疫监视和涉及MHC限制和T细胞识别的反应的组成部分。当通过MHC分子在T细胞上呈现在细胞表面上时，经过加工的抗原肽是由我们的免疫系统监测的身份标签。因此，抗原加工和表现会影响一个个体对感染生物的反应，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安装细胞毒性反应，导致靶细胞裂解。这种抗病毒机制通常涉及MHC I类分子，以结合从感染细胞中合成的病毒蛋白加工的肽，并将这些肽呈现为细胞毒性T淋巴细胞（CTL），这些肽可以消除感染细胞，从而消除新的病毒生产的潜在来源。因此，了解抗原加工的机制对于与各种病原体作斗争至关重要。为了使表位被T细胞识别，需要将外国抗原加工成具有适当尺寸的短肽，以形成与MHC分子的物理复合物。 I类抗原肽的前体主要由细胞质中的蛋白酶体产生，然后由与抗原加工（TAP）相关的转运蛋白转运到内质网（ER）的腔内。最近，ER内部的氨基肽酶（名为ERAAP）被确定为修剪肽前体并生成I类限制性表位的最终N末端的一个缺失的链接。我们最近在表达和净化活性ERAAP酶以及在初步结晶筛选中获得晶体的成功，这使得为这种关键酶提供高分辨率结构信息的令人兴奋的可能性。在这个探索性（R21）项目中，我们建议净化ERAAP的晶体学质量和数量，其子域和生化特征的复合物以及衍射质量质量的晶体，其长期目标是获得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，以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幻想的保护策略是必不可少的，因此对公共卫生至关重要。