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)：抗原处理是细胞介导的免疫监视和反应中不可或缺的一部分，涉及MHC限制和T细胞识别。经过处理的抗原肽，当由MHC分子呈现在细胞表面时，作为身份标签被我们的免疫系统监测。因此，抗原的处理和递呈影响个体对感染性生物的反应，并与疾病的易感性和自身免疫的发展有关。免疫系统清除病毒感染或恶性细胞的一种有效方法是启动细胞毒性反应，导致靶细胞溶解。这种抗病毒机制通常涉及MHC I类分子结合从感染细胞内合成的病毒蛋白加工而来的多肽，并将这些多肽呈递给细胞毒性T淋巴细胞(CTL)，细胞毒性T淋巴细胞可以清除感染的细胞，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。为了使表位被T细胞识别，外来抗原需要被加工成大小合适的短肽，以便与MHC分子形成物理复合体。I类抗原肽的前体主要由胞浆中的蛋白酶体产生，然后通过与抗原处理相关的转运蛋白(TAP)转运到内质网(ER)的管腔内。最近，内质网内的一种名为ERAAP的氨基肽酶被发现是一个缺失的环节，它负责修剪多肽前体并产生I类限制性表位的最终N末端。我们最近成功地表达和纯化了具有活性的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。在这个探索性项目(R21)中，我们建议提纯ERAAP、其亚结构域和络合物的结晶学质量和数量，用于生化表征和生长衍射质量的晶体，长期目标是获得ERAAP的高分辨率结构。与公众健康相关：在这项建议中获得的生化信息和衍射质量的晶体将使ERAAP的高分辨率结构分析能够增强我们对抗原处理机制的理解。这种结构信息对于制定生物防御保护战略是必要的，因此对公众健康至关重要。