BIOCHEMISTRY OF AN INTERNAL FATTY ACYLATION OF A PROTEIN

描述：蛋白质酰化是一种强大的翻译后信号。 机制。与转录中的N-肉豆蔻酸化类别形成对比 酰基蛋白质，纯蛋白质还没有被用来研究其他 一类酰基蛋白质，其内部残基是翻译后的 脂肪酰化。申请者的长期目标是研究 作为信号特征的蛋白质内酰化的生物化学 在哺乳动物蛋白质中的转导，如RAS。拟议的研究是一项 独一无二的机会，使用纯化的蛋白质，研究酶和 蛋白质内部酰化的生物化学，它导致深刻的 其功能发生了变化。细菌毒素系统被用作 蛋白质的内酰化。溶血素(HlyA)是RTX毒素的典型代表， 革兰氏阴性杆菌不同属分泌的细胞毒蛋白家族 结合和裂解特定哺乳动物细胞的细菌。毒素是 值得注意的是它们的异常激活，分泌方式，富含甘氨酸 非肽重复序列(RTX)，以及不同的细胞特异性。HlyA 起源于无毒的proHlyA，经翻译后修饰为 转酰基酶HlyC催化的长链脂肪酰化对HlyA的毒性作用。 据报道，酰基-ACP是体内脂肪的必需酰基供体 酰化反应。酰化不是分泌所必需的，但酰化是 使蛋白质有毒的单一因素。使用不同的重组 DNA载体HlyC和proHlyA已经被过度生产。激活 由HlyC催化的ProHlyA到HlyA的研究将与各种 用不同的放射性或化学试剂合成的酰基-ACP 为了确定反应的最佳条件， 优选底物、化学计量、动力学和机理。ProHylA+ *酰基-SACP-*HLYA+ACPSH。纯化的转酰基酶HlyC将被 特色化的。激活后HlyA特性的变化将是 傅里叶变换红外光谱分析(构象变化) 和流体动力光散射和荧光各向异性(在 聚集趋势)。 具有不同生物学功能和靶点的几个同源系统 细胞特异性使用溶血素方案产生毒性和 蛋白质分泌。例子包括分泌型根瘤菌。 NoDo蛋白(与HlyA同源)，感染豆类导致结瘤 HlyA本身在人类中具有重要的流行病学意义。溶血素 方案是一个反复出现的生物主题，使蛋白质有毒和 分泌具有感染性的细胞特异性蛋白。这个主题被用来 感染人类、植物和动物细胞以达到不同的目的 目标。

DESCRIPTION: Protein acylation is a powerful post-translational signaling mechanism. In contrast to the transcriptional N-myristoylation class of acyl proteins, pure proteins have not been available to study the other class of acyl proteins where internal residues are post-translationally fatty acylated. The applicant's long term objective is to study the biochemistry of protein internal acylation as a feature of signal transduction in mammalian proteins such as Ras. The proposed research is a unique opportunity to study, using purified proteins, the enzymology and biochemistry of an internal acylation of a protein which causes a profound change in its function. A bacterial toxin system is used as a model of protein internal acylation. Hemolysin (HlyA) typifies the RTX toxins, a family of cytotoxic proteins secreted by different genera of Gram negative bacteria which bind and lyse specific mammalian cells. The toxins are remarkable for their unusual activation, mode of secretion, glycine-rich nonapeptide repeats (RTX), and different cell specificities. HlyA originates as nontoxic proHlyA which is post-translationally modified to toxic HlyA by long chain fatty acylation catalyzed by HlyC, a transacylase. Acyl-ACP is reportedly the obligatory acyl donor for the internal fatty acylation. Acylation is not essential for secretion, but acylation is the single factor that renders the protein toxic. Using different recombinant DNA vectors, HlyC and proHlyA have been over produced. The activation of proHlyA to HlyA catalyzed by HlyC will be studied with a variety of acyl-ACPs which have been synthesized with different radioactive or fluorescent acyl groups in order to define the reaction optimum conditions, preferred substrate, stoichiometry, kinetics and mechanism. proHylA + *acyl-SACP---*HLYA + ACPSH. The purified transacylase, HlyC, will be characterized. Changes in HlyA characteristics upon activation will be examined by Fourier transform infrared spectroscopy (conformational changes) and hydrodynamic light scattering and fluorescence anistropy (change in aggregation tendencies). Several homologous systems with different biological functions and target cell specificities use the hemolysin scheme of generation of toxicity and protein secretion. Examples range from the secreted rhizobial bacterial protein NodO (homologous to HlyA) that infects legumes causing nodulation to HlyA itself which is epidemiologically important in humans. The hemolysin scheme is a recurring biological motif of rendering a protein toxic and secreting the infectious, cellular specific protein. This motif is used to infect human, plant, and animals cells en route to achieving different objectives.