FATTY ACYLATION OF INTERNAL RESIDUES OF A PROTEIN

蛋白质内部残基的翻译后脂肪酰化是改变蛋白质行为的一种强大但知之甚少的生物学机制。它在多种生理策略中具有重要作用，例如信号转导。本研究以大肠杆菌溶血素(HlyA)毒素的激活为模型来研究蛋白质的内酰化作用。致病性大肠杆菌产生无毒的proHlyA，通过翻译后将2个脂肪酰基添加到内部残基而使其有毒，导致蛋白质行为的非凡变化。酰基载体蛋白(ACP)是必需的酰基供体。HlyC是一种内部蛋白质酰基转移酶，它形成了酰基-HlyC中间体，证据表明它是酰基-组氨酸HlyC。使用精确和直接的分析，使用单独的亚克隆、纯化的蛋白质的反应是解释内部蛋白质酰基转移的生化机制(唯一被如此研究的)及其在改变蛋白质行为中的作用的独特机会。为了进一步了解蛋白质内部残基酰化的生物化学和相关的蛋白质行为变化，以及什么定义了蛋白质内部酰化位点，将追求以下具体目标：1.将使用定点突变分析、定点荧光和化学修饰来确定几个被证明在HlyC催化功能中重要的残基的作用，并将表征酰基-HlyC中间体。2.该反应可能是两个部分反应的总和，属于乒乓球动力学机制；第一个部分反应的可逆性已被证明。用所选择的放射性脂肪酰基标记HlyA，将显示第二部分反应的可逆性。3.研究亚克隆的proHlyA、含有两个或一个或另一个酰化位点的proHlyA-片段以及各自的酰化的HlyA片段的定点突变，以了解什么定义了内部蛋白质酰化位点，以及蛋白质在酰化过程中发生了什么变化。将观察到定点荧光探针的生物活性和荧光特性的变化。大肠杆菌溶血素是由不同的革兰氏阴性细菌产生的同源、相似活性的感染蛋白毒素家族中的一种。这些毒素具有显著的生物和细胞特异性。溶血素方案是一个重要的反复出现的生物基序，它使蛋白质有毒，并分泌具有感染性的细胞特异性蛋白质。深入了解这种独特的酰基转移的机制对于设计毒素激活抑制剂作为一种潜在的治疗方法具有重要的实际意义，因为在致病性大肠杆菌的严重感染中，抗生素治疗和随后的毒素释放会恶化临床结果。

Post-translational fatty acylation of internal protein residues is a powerful but poorly understood biological mechanism for altering a protein's behavior. It is important in diverse physiologic strategies, for example, signal transduction The activation of Escherichia coli hemolysin (HlyA) toxin is used in this proposal as a model to study protein internal acylation. Pathogenic E.coli produce nontoxic proHlyA which is made toxic by the post-translational addition of 2 fatty acyl groups to internal residues, causing an extraordinary change in protein behavior. Acyl-acyl carrier protein (ACP) is the obligatory acyl-donor. HlyC is the internal protein acyltransferase, which forms an acyl-HlyC intermediate, evidence suggests an acyl-histidyl HlyC. Using a precise and direct assay, the reaction employing separately subcloned, purified proteins is a unique opportunity to explain the biochemical mechanism of an internal protein acyltransfer (the only one to be so studied) and its role in changing protein behavior. The following specific aims will be pursued with the goals of further understanding the biochemistry of protein internal residue acylation and associated changes in protein behavior and what defines an internal protein acylation site: 1. Site- directed mutation analysis, site-directed fluorescence, and chemical modification will be used to define the roles of several residues shown to be important in HlyC's catalytic function, and the acyl-HlyC intermediate will be characterized. 2. The reaction is likely the sum of 2 partial reactions, a ping pong kinetic mechanism; the reversibility of the first has been shown. Using HlyA labeled with selected radioactive fatty acyl groups, the reversibility of the second partial reaction will be shown. 3. Site-directed mutations of subcloned proHlyA, proHlyA- fragments containing either both or one or the other acylation sites, HlyA, and the respective acylated HlyA fragments will be studied to learn what defines an internal protein acylation site and what changes a protein undergoes upon acylation. Alterations in biological activity and fluorescence characteristics of site-directed fluoroprobes will be observed. E. coli hemolysin typifies one of a family of homologous, similarly activated, infectious protein toxins produced by diverse Gram negative bacteria. The toxins have remarkable organism and cellular specificities. The hemolysin scheme is an important recurring biological motif of rendering a protein toxic and secreting the infectious cellular-specific protein. Insight into the mechanism of this unique acyltransfer is of practical importance in providing a rationale for design of inhibitors of toxin activation as a potential therapeutic approach to severe infections with pathogenic E. coli where antibiotic therapy and subsequent toxin release worsen the clinical outcome.