Complement Convertase: Assembly, Function and Regulation

描述（由申请人提供）：补体系统是先天性和获得性免疫的关键参与者。补体标记免疫清除或细胞溶解的感染因子，并形成炎症反应的焦点。此外，补体促进抗原定位于脾和淋巴结，在那里它降低了特异性B细胞活化的阈值。补体也可以是人类疾病如抗体介导的自身免疫、免疫复合物沉积综合征、缺血再灌注损伤和超急性移植排斥中组织损伤的主要原因。 补体的几乎所有生物学后果都需要C3（一种丰富的血清糖蛋白）的酶促裂解。裂解C3的酶C3转化酶是补体激活途径中的主要参与者，并且以两种结构和功能上同源的形式存在，即旁路途径（AP）C3转化酶（C3 bBb）和经典途径（CP）C3转化酶（C4 bC 2a）。几种结构上相关的蛋白质，CR 1，C4 BP和H因子，抑制不适当的补体激活（例如在自身组织上）。它们的作用是促进活性转化酶的不可逆解离，这一过程称为衰变加速。相反，血清蛋白备解素可以部分稳定靶表面上的C3 bBb。这项工作的长期目标是创造新的方法来治疗控制补体。我们的策略是阐明补体激活中的关键酶C3转化酶的独特生化特征，并确定有前途的治疗靶点。开发了用于分析AP C3转化酶C3 bBb和C3 bBbP的组装、稳定化和衰变加速的简单测定法：用C3 B包被微量滴定威尔斯孔，与液相因子B、因子D、二价阳离子以及在某些情况下的备解素一起孵育;通过ELISA检测复合物。采用基于ELISA的测定法，通过定点诱变产生因子B、CR 1和CR 1突变体，鉴定了许多可能的活性位点，并提出了几个关键假设。为了检验这些假设，提出了具有以下具体目标的研究： 1)开发转化酶组装及其丝氨酸蛋白酶结构域活化的模型; 2)确定备解素如何稳定AP转化酶; 3)阐明加速衰变的机制。

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.