Structural studies on an aminopeptidase inside the endoplasmic reticulum

内质网内氨肽酶的结构研究

基本信息

批准号：
7643263
负责人：
Hwai-Chen Guo
金额：
$ 8.13万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7643263
关键词：
Aminopeptidase Antigen Presentation Pathway Antigens Antiviral Agents Autoimmunity Baculoviruses Binding Biochemical C-terminal Cell surface Cells Complex Crystallization Cytolysis Cytosol Cytotoxic T-Lymphocytes Development Disease Endoplasmic Reticulum Enzymes Epitopes Failure Goals Histocompatibility Antigens Class I Immune system Immunologic Surveillance In Vitro Individual Infectious Agent Insecta Length Link Mediating Molecular Monitor Names Peptide Hydrolases Peptide/MHC Complex Peptides Phase Predisposition Process Production Proteins Public Health Reaction Recombinant Proteins Research Project Grants Resolution Source Structure Substrate Specificity System T-Lymphocyte Viral Viral Proteins Virus antigen processing antigenic peptide transporter base biodefense cancer cell cytotoxic experience fight against improved multicatalytic endopeptidase complex novel pathogen public health relevance response scale up success

项目摘要

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 foreign antigenic peptides to cytotoxic T lymphocytes (CTLs), which can eliminate virus-infected self-cells and thus eliminate potential sources of new viral production. Therefore, understanding the mechanisms of antigen processing is critical to fight against various pathogens. For an epitope to be recognized by T cells, foreign antigens need to be cut 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 ERAP1, 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 ERAP1, and obtaining crystals of its C-terminal domain (C-dom) in the preliminary crystallization screens, have brought about the exciting possibility of providing high-resolution structural information for this critical enzyme. In this Small Research Project (R03) application, we propose to purify crystallographic quality and quantity of ERAP1 C-dom and complexes for biochemical characterization and high-resolution crystal structure determination. PUBLIC HEALTH RELEVANCE: Crystallographic quality and quantity of ERAP1 C-dom, N-dom, and full-length protein obtained in this proposal will enable biochemical and structural studies of ERAP1 to enhance our understanding of the mechanisms of antigen processing. This 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，并将这些外抗原肽与细胞毒性T淋巴细胞（CTL）呈现，从而消除病毒感染的自我细胞，从而消除新的病毒式生产。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，需要将外抗原切成适当尺寸的短肽，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（称为ERAP1）被确定为修剪肽前体并生成I类限制性表位的最终N末端的缺失链接。我们最近在表达和净化活动的ERAP1方面的成功，并在初步的结晶筛选中获得其C末端结构域（C-DOM）的晶体，带来了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。在这个小型研究项目（R03）应用中，我们建议净化晶体学质量和ERAP1 C-DOM和复合物的数量，以进行生化特征和高分辨率晶体结构测定。公共卫生相关性：在本提案中获得的ERAP1 C-DOM，N-DOM和全长蛋白的晶体学质量和数量将使ERAP1的生化和结构研究能够增强我们对抗原处理机制的理解。该信息对于制定生物幻想的保护策略是必要的，因此对公共卫生至关重要。