Complement Convertase: Assembly, Function and Regulation

补体转化酶：组装、功能和调节

• 批准号: 6616462
• 负责人: DENNIS EMIL HOURCADE
• 金额: $ 22.95万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616462
• 关键词: active sites; complement; complement pathway; enzyme activity; enzyme linked immunosorbent assay; hydrolase; molecular assembly /self assembly

DESCRIPTION (provided by applicant): The complement system is a critical participant in both innate and acquired immunity. Complement marks infectious agents for immune clearance or cell lysis and it forms a focal point for inflammatory reactions. Moreover, complement facilitates antigen localization to the spleen and lymph nodes, where it lowers the threshold for the activation of specific B cells. Complement can also be a principal cause of tissue damage in human diseases such as antibody-mediated autoimmunity, immune complex deposition syndromes, ischemic reperfusion injury, and hyperacute graft rejection. Nearly all the biological consequences of complement require the enzymatic cleavage of C3, an abundant serum glycoprotein. The enzymes that cleave C3, the C3 convertases, are major players in the complement activation pathways and occur in two structurally and functionally homologous forms, the alternative pathway (AP) C3 convertase (C3bBb) and the classical pathway (CP) C3 convertase (C4bC2a). Several structurally related proteins, DAF, CR1, C4BP, and factor H, inhibit inappropriate complement activation (e.g. on self-tissue). They act to promote the irreversible dissociation of active convertases, a process known as decay acceleration. In contrast, the serum protein properdin can partially stabilize C3bBb on target surfaces. The long-range goal of this work is to create new approaches to the therapeutic control of complement. Our strategy is to elucidate the unique biochemical features of the C3 convertases, the key enzymes in complement activation, and identify promising therapeutic targets. A simple assay was developed for analyzing the assembly, stabilization, and decay acceleration of the AP C3 convertases C3bBb and C3bBbP: Microtiter wells were coated with C3b, incubated with fluid phase factor B, factor D, divalent cation and, in some cases, properdin; complexes were detected by ELISA. Employment of the ELISA-based assay with panels of factor B, DAF and CR1 mutants generated by site-directed mutagenesis has led to the identification of a number of possible active sites and the proposal of several key hypotheses. To test these hypotheses, studies are proposed with the following specific aims: 1) Develop a model for convertase assembly and the activation of its serine protease domain; 2) Determine how properdin stabilizes AP convertases; 3) Elucidate the mechanisms of decay acceleration.

描述（由申请人提供）：补体系统是先天和获得免疫的关键参与者。补体标志着免疫清除或细胞裂解的感染剂，它构成了炎症反应的焦点。此外，补体促进抗原定位到脾脏和淋巴结，在此降低了特定B细胞激活的阈值。补体也可能是人类疾病中组织损伤的主要原因，例如抗体介导的自身免疫，免疫复合物沉积综合征，缺血性再灌注损伤和超急性移植抑制。 补体的几乎所有生物学后果都需要C3（丰富的血清糖蛋白）的酶促切割。裂解C3（C3转化酶）是补体激活途径中的主要参与者，并以两个结构和功能同源形式出现，替代途径（AP）C3转化酶（C3BBB）和经典途径（CP）C3 Convertase（C4BC2A）。几种与结构相关的蛋白DAF，CR1，C4BP和因子H抑制了不适当的补体激活（例如，在自组织上）。它们的作用是促进活性转化酶的不可逆解离，这一过程称为衰减加速度。相比之下，血清蛋白适用于靶表面上的C3BBB可以部分稳定。这项工作的远距离目标是为补充的治疗控制创造新的方法。我们的策略是阐明C3转化酶的独特生物化学特征，补体激活中的关键酶，并确定有希望的治疗靶标。开发了一个简单的测定，用于分析AP C3转化酶C3BBB和C3BBB的组装，稳定和衰减加速度：与C3B涂覆的微量滴定孔，并与流体相位因子B，因子D，Divalent阳离子，Divalent阳离子，在某些情况下，Poredin； ELISA检测到复合物。使用位置定向诱变产生的因子B，DAF和CR1突变体的面板的基于ELISA的测定，导致鉴定了许多可能的活性位点，并提出了几个关键假设的建议。为了检验这些假设，提出了以下具体目的的研究： 1）开发转化酶组装的模型及其丝氨酸蛋白酶域的激活； 2）确定适当稳定AP转化酶的正确性； 3）阐明衰减加速度的机制。