Biochemistry of Protein Internal Residue Acylation

描述(由申请人提供)：内部蛋白质残基的翻译后脂肪酰化是一种强大的生物学设计，可以最终并在某些情况下可逆地改变蛋白质的行为。本文以大肠杆菌溶血素(HlyA)毒素的激活为模型，研究蛋白质的内酰化作用。致病性大肠杆菌产生无毒的proHlyA，翻译后将2个脂肪酰基添加到特定的内部赖氨酸残基上，使其有毒，这是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，在最终酰基转移到proHlyA之前形成酰基-HlyC中间体。酰化区域的选择小肽模拟物抑制了酰基-HlyC的酰基转移，提示识别特征。通过灵敏、直接的分析和纯蛋白质，该反应能够对内部残基蛋白质酰化和蛋白质行为的修饰进行独特的生物化学研究。利用定点突变分析/荧光分析、图像分析和等温滴定量热法等方法，以下3个特定目标将阐明内部蛋白质酰化的生物化学及其相关的变化，以及如何识别内部蛋白质酰化部位/区域：1.在反应中哪里是酰基碳链长度的关键--在酰基-HlyC的形成或从酰基-HlyC向ProHlyA的转移中？2.酰化部位附近的哪些氨基酸对酰化区域的识别至关重要？3.是什么使这两个酰化区域作为酰化底物的效率不同？大肠杆菌溶血素是潜在生物恐怖分子名单上的一种病原体，它是由各种革兰氏阴性细菌分泌的一种同源、类似活性的感染蛋白毒素家族的典型代表。这些毒素具有显著的生物和细胞特异性。HlyA的毒性明确地仅依赖于酰化作用。深入了解这种独特的酰基转移-毒性-激活的机制具有基本的科学意义和治疗前景。如上所述，大肠杆菌的毒性取决于毒素的激活。致病性大肠埃希氏菌感染不能用抗生素治疗；没有特效药。阐明毒素激活的机制将有助于合理设计特定的毒素激活抑制剂。本文中描述的停止激活的多肽有助于理解激活，具有治疗适应的潜力，为这种可怕的感染提供合理的治疗方法。

DESCRIPTION (provided by applicant): Post-translational fatty acylation of internal protein residues is a powerful, biological design for definitively and in some instances reversibly altering protein behavior. The activation of Escherichia coli hemolysin (HlyA) toxin is used here as a model to study protein internal acylation. Pathogenic E. coli produce nontoxic proHlyA that is made toxic by post-translational addition of 2 fatty acyl groups to specific internal lysine residues, an extraordinary change in protein behavior. Acyl-acyl carrier protein (ACP) is the obligatory acyl donor. HlyC is the internal protein acyl-transferase that forms acyl-HlyC intermediate prior to ultimate acyl transfer to proHlyA. Transfer of acyl-HlyC's acyl group is inhibited by selected small peptide mimetics of acylation regions suggesting recognition features. With sensitive, direct assays and pure proteins, the reaction enables unique study of the biochemistry of internal residue protein acylation and modification of protein behavior. Using site-directed mutation analysis/fluorescence, image analysis, and isothermal titration calorimetry, among other methods, the following 3 specific aims will clarify the biochemistry of internal protein acylation, its associated changes, and how an internal protein acylation site/region is recognized: 1. Where in the reaction is acyl group carbon chain length crucial-in the formation of acyl-HlyC or in the transfer from acyl-HlyC to proHlyA? 2. What amino acids proximal to acylation sites are crucial to recognition by acyl-HlyC as an acylation region? 3. What makes the two acylation regions differ in their efficacies as acylation substrates? E. coli hemolysin, a pathogen on the list of potential bioterrorism agents, typifies one of a family of homologous, similarly activated, infectious protein toxins secreted by diverse Gram-negative bacteria. The toxins have remarkable organism and cellular specificities. HlyA's toxicity is unequivocally dependent solely upon acylation. Insight into the mechanism of this unique acyltransfer-toxic-activation has basic scientific significance and therapeutic promise. E. coli toxicity depends upon activation of the toxin as described above. Pathogenic E. coli infection cannot be treated with antibiotics; there is no specific treatment. Elucidation of the mechanism of toxin activation will enable logical design of specific inhibitors of toxin activation. The peptides that stop activation described herein to help to understand activation have the potential for therapeutic adaptation to provide a rational therapy for this horrible infection.