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，可以明确地，在某些情况下可逆地改变蛋白质行为。本文以大肠杆菌溶血素（HlyA）毒素的激活为模型，研究蛋白质内部酰化。致病性大肠杆菌产生无毒的proHlyA，通过翻译后在特定的内部赖氨酸残基上添加2个脂肪酰基而产生毒性，这是蛋白质行为的一个非凡变化。酰基酰基载体蛋白（ACP）是必需的酰基供体。HlyC是蛋白质内部的酰基转移酶，在最终酰基转移到proHlyA之前形成酰基-HlyC中间体。酰基- hlyc的酰基转移被选定的酰化区域的小肽模拟物抑制，表明识别特征。通过灵敏、直接的检测和纯净的蛋白质，该反应能够独特地研究内部残基蛋白酰化和蛋白质行为修饰的生物化学。利用定点突变分析/荧光、图像分析和等温滴定量热法等方法，以下3个特定目的将阐明内部蛋白质酰化的生物化学，其相关变化，以及如何识别内部蛋白质酰化位点/区域：在反应中，酰基碳链长度在形成酰基- hlyc或从酰基- hlyc到proHlyA的转移中是至关重要的？2. 酰化位点附近的哪些氨基酸对酰基- hlyc识别为酰化区域至关重要？3. 是什么使得这两个酰化区域作为酰化底物的功效不同？大肠杆菌溶血素是潜在生物恐怖主义制剂清单上的一种病原体，是由多种革兰氏阴性菌分泌的同源、类似激活的传染性蛋白毒素家族的一种。毒素具有显著的生物体和细胞特异性。HlyA的毒性完全依赖于酰化。深入了解这种独特的酰基转移毒性激活机制具有基本的科学意义和治疗前景。大肠杆菌的毒性取决于上述毒素的活化。致病性大肠杆菌感染不能用抗生素治疗；没有具体的治疗方法。毒素活化机制的阐明将使毒素活化特异性抑制剂的逻辑设计成为可能。本文描述的阻止激活的肽有助于理解激活，具有治疗适应的潜力，为这种可怕的感染提供合理的治疗。

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.