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转导 机制 与转录的N-豆蔻酰化类相反， 酰基蛋白，纯蛋白还没有可用于研究的其他 一类酰基蛋白质，其内部残基是后降解的 脂肪酰化。 申请人的长期目标是研究 作为信号特征的蛋白质内部酰化的生物化学 哺乳动物蛋白如Ras的转导。拟议的研究是一个 独特的机会，研究，使用纯化的蛋白质，酶学和 蛋白质内部酰化的生物化学， 改变其功能。 细菌毒素系统被用作 蛋白质内部酰化。 溶血素（HlyA）代表RTX毒素， 不同属革兰氏阴性菌分泌的细胞毒性蛋白家族 结合并溶解特定哺乳动物细胞的细菌。 毒素是 以其不寻常的激活、分泌方式、富含甘氨酸 九肽重复序列（RTX）和不同的细胞特异性。 HlyA 起源于无毒的proHlyA，其经过后修饰， 毒性HlyA通过由HlyC（一种转酰酶）催化的长链脂肪酰化。 据报道，酰基-ACP是内部脂肪酸的强制性酰基供体。 酰化反应。 酰化不是分泌所必需的，但酰化是分泌的关键。 使蛋白质有毒的单一因素。 使用不同的重组 DNA载体HlyC和proHlyA已经过度生产。 的激活 由HlyC催化的proHlyA到HlyA将用多种方法进行研究。 酰基-ACP已经用不同的放射性或 荧光酰基为了确定反应的最佳条件， 优选的底物、化学计量、动力学和机理。 proHylA + * 酰基-SACP-*HLYA + ACPSH。 纯化的转酰酶HlyC将被纯化。 表征了 激活后HlyA特性的变化将是 通过傅里叶变换红外光谱法检查（构象变化） 以及流体动力学光散射和荧光各向异性（在光散射中的变化）。 聚集趋势）。 具有不同生物学功能和靶点的几种同源系统 细胞特异性使用溶血素产生毒性的方案， 蛋白质分泌 例子包括分泌的根瘤菌 NodO蛋白（与HlyA同源），感染豆科植物，引起结瘤， HlyA本身在人类流行病学上很重要。 溶血素 该方案是一个反复出现的生物学基序，使蛋白质有毒， 分泌感染性的细胞特异性蛋白质 这个主题是用来 感染人类、植物和动物细胞， 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.