Structure and Function of ADAMTS13 Metalloprotease

ADAMTS13 金属蛋白酶的结构和功能

基本信息

批准号：
7152582
负责人：
X. Long Zheng
金额：
$ 31.48万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-12-01 至 2009-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7152582
关键词：
ADAMTS Acids Active Sites Address Adult Affinity Alanine Anabolism Apical Aspartic Acid Autoantibodies Basolateral Sorting Signal Binding Binding Sites Biological Biological Assay Blood Circulation Cellular Morphology Chimeric Proteins Cleaved cell Clinical Daily Deletion Mutagenesis Disintegrin Domain Dissociation Endopeptidases Enzymes Exhibits Genes Glutamine Goals Green Fluorescent Proteins Hemostatic function Idiopathic Thrombocytopenic Purpura Immunoassay Indium Integrin Binding Kinetics Label Length Link Localized MDCK cell Macroglobulins Metalloproteases Mutagenesis Oligosaccharides Outcome Patients Peptide Hydrolases Peptides Plasma Plasma Exchange Point Mutation Prevention Proteins Purpura Radiolabeled Recombinants Regulation Research Research Personnel Role Signal Transduction Site Sorting - Cell Movement Specificity Structure Surface Plasmon Resonance Syndrome Thrombocytopenic Purpura Thrombosis Thrombospondin 1 Thrombotic Thrombocytopenic Purpura Time Western Blotting Zinc analog base domain mapping mutant programs radioligand radiotracer research study tool von Willebrand Factor

项目摘要

DESCRIPTION (provided by applicant): The catastrophic complications due to deficiency in plasma metalloprotease (ADAMTS13) highlight the biological importance of this protease in regulation of hemostasis and prevention of thrombosis in microvascular circulation that occurs in the thrombotic thrombocytopenic purpura (TTP) syndrome. Identification of the gene encoding ADAMTS13 metalloprotease and determination of the structure elements in the encoded protein have opened a new era in understanding of the roles of a zinc metalloprotease in regulation of hemostasis and thrombosis, and provided the essential tools to address the fundamental (patho)biological questions concerning ADAMTS13 including its biosynthesis, activation, substrate recognition, intracellular sorting and autoantibody interaction. The goals of the proposed research are: Specific Aim 1. To determine ADAMTS13 substrate recognition by 1) analyzing the proteolytic activity of wild type ADAMTS13 and various mutants of ADAMTS13 toward von Willebrand factor and its analog, VWF73; 2) determining the binding kinetics between VWF or VWF73 and wild type or mutant ADAMTS13 by radioligand binding and surface plasmon resonance assays. Specific Aim 2. To determine intracellular sorting of ADAMTS13 by 1) determining the polarity of full-length and mutant ADAMTS13 secretion in MDCK cells; 2) determining the sorting signal and mechanisms by which ADAMTS13 is sorted; 3) determining the effect of point mutations or truncations of ADAMTS13 gene found in patients with congenital TTP on the polarity of ADAMTS13 secretion. Specific Aim 3. To determine the domains (or sites) of ADATMTS13 to which anti-ADAMTS13 autoantibodies bind and the kinetic parameters (affinity and specificity) of interaction between ADAMTS13 metalloprotease and anti-ADAMTS13 autoantibodies, and to correlate the clinical course and outcome to the distinct type of anti-ADAMTS autoantibodies identified in patients with TTP.

描述（由申请人提供）：由于血浆金属蛋白酶（ADAMTS13）缺乏而导致的灾难性并发症凸显了该蛋白酶在调节止血和预防血栓性血小板减少性紫癜（TTP）综合征中发生的微血管循环血栓形成方面的生物学重要性。编码 ADAMTS13 金属蛋白酶的基因的鉴定和所编码蛋白质中结构元件的测定，开启了了解锌金属蛋白酶在止血和血栓形成调节中的作用的新纪元，并为解决根本（病理）问题提供了必要的工具。有关 ADAMTS13 的生物学问题，包括其生物合成、激活、底物识别、细胞内分选和自身抗体相互作用。拟议研究的目标是：具体目标 1. 通过以下方式确定 ADAMTS13 底物识别： 1) 分析野生型 ADAMTS13 和 ADAMTS13 的各种突变体对冯维勒布兰德因子及其类似物 VWF73 的蛋白水解活性； 2)通过放射性配体结合和表面等离振子共振测定法确定VWF或VWF73与野生型或突变体ADAMTS13之间的结合动力学。具体目标 2. 通过 1) 确定 MDCK 细胞中全长和突变 ADAMTS13 分泌的极性来确定 ADAMTS13 的细胞内分选； 2)确定ADAMTS13的排序信号和排序机制； 3)确定先天性TTP患者中发现的ADAMTS13基因点突变或截短对ADAMTS13分泌极性的影响。具体目标 3. 确定抗 ADAMTS13 自身抗体结合的 ADATMTS13 结构域（或位点）以及 ADAMTS13 金属蛋白酶和抗 ADAMTS13 自身抗体之间相互作用的动力学参数（亲和力和特异性），并将临床病程和结果与在 TTP 患者中发现的独特类型的抗 ADAMTS 自身抗体。