HLA-Releasing Metalloproteinase in Allograft Rejection

同种异体移植排斥中 HLA 释放金属蛋白酶

• 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
• 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

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类的释放是由崩解素和金属蛋白酶家族成员ADAM17介导的。内皮细胞（EC）与异体T细胞共培养可上调特异性激活标记物以及adam17的表达和活性。这种激活是由干扰素驱动的，并在EC释放可溶性MHC I类蛋白时达到高潮。然而，至少有一种与ADAM17不同的其他金属蛋白酶完全能够释放可溶性MHC i类，其活性可能受细胞因子以组织特异性方式调节。用我们的mAb筛选人类白血病表达文库，阻断可溶性MHC类I的释放，鉴定出一种功能未知的新蛋白BC036469。这种无处不在表达的蛋白可能通过介导特异性酶/底物相互作用参与可溶性MHC I类释放机制，其功能可能受到不同组织中细胞特异性细胞因子的调节。三个独立的目标旨在解决这些问题。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MHC I类的细胞因子诱导的金属蛋白酶。其次，BC036469蛋白的功能将通过过表达野生型和缺失型突变体，以及用特异性siRNA破坏内源蛋白的表达来确定。最后，我们将确定α 3和MHC I类跨膜结构域内生产酶/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相关抑制模型中使用跨体延迟型超敏反应试验进行测试。