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Molecular Analysis of CD8, MHC Class I Interaction

CD8、MHC I 类相互作用的分子分析

基本信息

批准号：
8210897
负责人：
Paula B. Kavathas
金额：
$ 34.11万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8210897
关键词：
Adaptor Signaling Protein Adoptive Immunotherapy Affect Affinity Agonist Alternative Splicing Amino Acids Binding Biological Assay CD8-Positive T-Lymphocytes CD8B1 gene CDR1 gene Cell Line Cell Separation Cells Chemicals Complex Cytomegalovirus Cytoplasmic Tail Cytotoxic T-Lymphocytes Development Escherichia coli Extracellular Domain Failure Fluorescence Resonance Energy Transfer Funding Genes Grant Human Human Cell Line Human Identifications Hybridomas Immunoglobulin Domain In Vitro Individual Kinetics Knock-out Label MHC Class I Genes Maps Measurement Membrane Proteins Memory Messenger RNA Molecular Molecular Analysis Mus Mutagenesis Mutation NMR Spectroscopy Outcome Peptide/MHC Complex Peptides Phosphorylation Plasmids Play Population Distributions Positioning Attribute Production Protein Isoforms Protein Tyrosine Kinase Proteins RNA Splicing Regulation Role Signal Transduction Solutions Surface Surface Plasmon Resonance System T cell response T-Cell Activation T-Cell Development T-Cell Receptor T-Lymphocyte TCR Activation Testing Transfection Ubiquitination Variant Viral Work base cell killing cytokine design dimer insight killings melanoma mutant neoplastic cell pathogen protein protein interaction public health relevance receptor internalization research study small hairpin RNA tumor

项目摘要

DESCRIPTION (provided by applicant): The CD8 coreceptor is a critical molecule for T cell activation and development forming a trimolecular complex with the T cell receptor, and peptide-MHC class I (pMHC1). As the CD8ab heterodimer is 100 fold more efficient than CD8aa as a coreceptor, we are studying the role of CD8b in coreceptor function. We identified critical residues on the CDR-like loops of the immunoglobulin-like domain CD8b protein required for binding to pMHC1 as well as mutants that led to enhanced binding. Based on our studies we proposed that CD8ab has two binding modes for interaction with MHC class I. Understanding the molecular basis for CD8ab-pMHCI interaction is hampered due to a lack of structural information. In aim 1, we will determine structurally how CD8ab and pMHCI interact by NMR spectroscopy and test our hypothesis that there are two binding modes. In aim 2, we will further characterize the murine CD8b enhancing mutants by making affinity measurements with surface plasmon resonance studies and will compare the enhancement of coreceptor activity with agonist, weak agonist, and antagonist peptides using FRET analysis of CD8b-TCR interaction. In addition, we will identify human CD8b enhancing mutants using an approach that involves random mutagenesis at individual amino acid residues in CDR- loops, transfection of plasmids into COS7 cells for transient expression, and FACS sorting for cells expressing CD8b mutants with enhanced binding to an HLA class I tetramer. The human CD8b protein has multiple isoforms with different cytoplasmic tails arising from alternative splicing, unlike murine CD8b. We found differential expression at the level of mRNA of the four human CD8b splice variants (M1-4) during development, after activation, and in memory subsets. and surface expression of one of the isoforms (M-2) was regulated by ubiquitination. We hypothesize that these isoforms contribute to differential regulation of CD8 T cell responses in humans. In aim 3, we will study functional differences between the isoforms and determine the molecular basis for those differences. Different murine and human cell lines will be used including human CD8 T cells from an anti-melanoma clone and an anti-viral cytomegalovirus specific line. Motifs within the cytoplasmic tails suggesting molecular mechanisms, e.g. phosphorylation, dileucine, Grb2 binding, will be studied by creating mutations in the motifs and performing functional assays, association with adaptor proteins, coreceptor internalization, and signal transduction. The outcome of the proposed studies will provide insights into a critical protein-protein interaction, CD8-pMHC1, for the CD8 cytotoxic T cells that kill tumor cells and cells infected with intracellular pathogens. The information we obtain will serve as a basis for designing an "optimized"CD8b with enhanced binding to pMHC1 and/or optimal signaling that potentially could be used for adoptive cellular immunotherapy against tumors. PUBLIC HEALTH RELEVANCE: The long-term objective is to isolate an enhanced human CD8b mutant, and to determine the functional significance of human CD8b splice variants with different cytoplasmic tails in order to design an optimized CD8b protein that potentially can be used for adoptive cellular immunotherapy.

描述（由申请人提供）：CD 8辅助受体是T细胞活化和发育的关键分子，与T细胞受体和肽-MHC I类（pMHC 1）形成三分子复合物。由于CD 8ab异源二聚体作为辅助受体的效率是CD 8aa的100倍，因此我们正在研究CD 8b在辅助受体功能中的作用。我们鉴定了与pMHC 1结合所需的免疫球蛋白样结构域CD 8b蛋白的CDR样环上的关键残基以及导致结合增强的突变体。基于我们的研究，我们提出CD 8ab与MHC I类分子有两种结合模式。由于缺乏结构信息，理解CD 8ab-pMHCI相互作用的分子基础受到阻碍。在目标1中，我们将通过NMR光谱确定CD 8ab和pMHCI在结构上如何相互作用，并测试我们的假设，即存在两种结合模式。在目标2中，我们将通过表面等离子体共振研究进行亲和力测量来进一步表征小鼠CD 8b增强突变体，并将使用CD 8b-TCR相互作用的FRET分析来比较激动剂、弱激动剂和拮抗剂肽对辅助受体活性的增强。此外，我们将使用一种方法鉴定人CD 8b增强突变体，该方法涉及在CDR环中的单个氨基酸残基处的随机诱变，将质粒转染到COS 7细胞中用于瞬时表达，以及对表达与HLA I类四聚体结合增强的CD 8b突变体的细胞进行FACS分选。与鼠CD 8b不同，人CD 8b蛋白具有多种同种型，其具有由选择性剪接产生的不同胞质尾。我们发现，在mRNA水平的差异表达的四个人CD 8b剪接变异体（M1-4）在发展过程中，激活后，和记忆子集。其中一种亚型（M-2）的表面表达受泛素化调控。我们假设这些亚型有助于人类CD 8 T细胞反应的差异调节。在目标3中，我们将研究异构体之间的功能差异，并确定这些差异的分子基础。将使用不同的鼠和人细胞系，包括来自抗黑素瘤克隆和抗病毒巨细胞病毒特异性系的人CD 8 T细胞。细胞质尾部内的基序表明分子机制，例如磷酸化，双亮氨酸，Grb 2结合，将通过在基序中创建突变并进行功能测定，与衔接蛋白，辅助受体内化和信号转导的关联来研究。拟议的研究结果将提供深入了解一个关键的蛋白质-蛋白质相互作用，CD 8-pMHC 1，为CD 8细胞毒性T细胞，杀死肿瘤细胞和细胞内病原体感染的细胞。我们获得的信息将作为设计“优化的“CD 8b的基础，该CD 8b具有增强的与pMHC 1的结合和/或最佳的信号传导，其可能用于针对肿瘤的过继细胞免疫疗法。公共卫生相关性：长期目标是分离增强的人CD 8b突变体，并确定具有不同胞质尾的人CD 8b剪接变体的功能意义，以设计可能用于过继性细胞免疫治疗的优化的CD 8b蛋白。