HLA-Releasing Metalloproteinase in Allograft Rejection

同种异体移植排斥中 HLA 释放金属蛋白酶

基本信息

批准号：
7373536
负责人：
YURI BUSHKIN
金额：
$ 32.95万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7373536
关键词：
Adam15 gene Address Allogenic Allografting Alternative Splicing Antigen Presentation Avidity Biological Assay CD8B1 gene Cell Line Cells Chimera organism Chimeric Proteins Class Cleaved cell Coculture Techniques Complex Cytomegalovirus DNA Microarray Chip DNA Microarray format Data Delayed Hypersensitivity Disintegrins Endothelial Cells Enzymes Expression Library Family member Fibroblasts Genes Goals Granulocyte-Macrophage Colony-Stimulating Factor HLA-A2 Antigen Homologous Gene Human Immune Infection Interferon Type II Interferons Link Major Histocompatibility Complex Measures Mediating Metalloproteases Microarray Analysis Modeling Monoclonal Antibodies Mus Pathway interactions Peptides Peripheral Blood Mononuclear Cell Physiological Process Protein Overexpression Proteins Regulation Role Screening procedure Site Small Interfering RNA Stimulus Substrate Interaction Surface System T-Lymphocyte Testing Tissues Transmembrane Domain Transplantation Transplantation Tolerance blastomere structure cytokine design enzyme activity enzyme substrate extracellular in vivo inhibitor/antagonist leukemia monocyte mutant novel response

项目摘要

DESCRIPTION (provided by applicant): We have previously described the metalloproteinase-mediated pathway of soluble MHC class I release and proposed its role in transplantation. We found that the release of soluble MHC class I is mediated by a disintegrin and metalloprotease family member, ADAM17. Endothelial cells (EC) co-cultured with allogeneic T cells up-regulate specific activation markers and both the expression and activity of ADAM 17. This activation is driven by interferon-gamma and culminates in the release of soluble MHC class I proteins by EC. However, at least one other metalloproteinase distinct from ADAM17 is fully capable of releasing soluble MHC class I. Its activity may be regulated by cytokines in a tissue-specific manner. Screening of a human leukemia expression library with our mAb that blocks the release of soluble MHC class I led to identification of a novel protein BC036469 with yet unknown function. This ubiquitously expressed protein may participate in the mechanism of soluble MHC class I release by mediating specific enzyme/substrate interactions and its function may be regulated by cell-specific cytokines in different tissues. Three independent aims are designed to address these questions. First, we will identify cytokine-inducible metalloproteinases capable of processing MHC class I by using a panel of ADAM-deficient cell lines and by DNA microarray analysis. Second, the function of BC036469 protein will be determined by overexpressing wild type and deletion mutants, and by disrupting expression of the endogenous protein with specific siRNA. Finally, we will determine the sites required for productive enzyme/substrate interactions within alpha 3 and transmembrane domains of MHC class I and test the ability of specific peptides to inhibit the interaction. The predicted role of soluble MHC class I in antigen presentation will be tested using the trans-vivo delayed-type hypersensitivity assay in the linked suppression model of immune regulation by HLA-A2- restricted CD8 low avidity T regulator cells controlling transplantation tolerance.

描述（由申请人提供）：我们之前已经描述了金属蛋白酶介导的可溶性MHC I类释放途径并提出了其在移植中的作用。我们发现可溶性 MHC I 类的释放是由解整合素和金属蛋白酶家族成员 ADAM17 介导的。与同种异体 T 细胞共培养的内皮细胞 (EC) 上调特定激活标记以及 ADAM 17 的表达和活性。这种激活由干扰素 γ 驱动，最终导致 EC 释放可溶性 MHC I 类蛋白。然而，至少一种不同于 ADAM17 的其他金属蛋白酶完全能够释放可溶性 MHC I 类。其活性可能以组织特异性方式受到细胞因子的调节。使用我们的可阻断可溶性 MHC I 类释放的 mAb 筛选人白血病表达文库，鉴定出一种功能尚不清楚的新型蛋白质 BC036469。这种普遍表达的蛋白质可能通过介导特定的酶/底物相互作用参与可溶性 MHC I 类释放机制，并且其功能可能受到不同组织中细胞特异性细胞因子的调节。设计了三个独立的目标来解决这些问题。首先，我们将通过使用一组 ADAM 缺陷细胞系和 DNA 微阵列分析来鉴定能够处理 I 类 MHC 的细胞因子诱导型金属蛋白酶。其次，BC036469 蛋白的功能将通过过表达野生型和缺失突变体以及通过用特异性 siRNA 破坏内源蛋白的表达来确定。最后，我们将确定 MHC I 类的 α 3 和跨膜结构域内有效酶/底物相互作用所需的位点，并测试特定肽抑制相互作用的能力。可溶性 MHC I 类在抗原呈递中的预测作用将在 HLA-A2 限制性 CD8 低亲和力 T 调节细胞控制移植耐受的免疫调节连锁抑制模型中使用体内迟发型超敏反应测定进行测试。