Activation of PI-Specific Phospholipase C Enzymes

描述（由申请人提供）： 磷脂酰肌醇特异性磷脂酶C酶催化磷酸肌醇裂解为二酰基甘油（蛋白激酶C的第二信使激活剂）和肌醇磷酸（在细菌PI-PLC的情况下为环状1，2-肌醇磷酸，而在哺乳动物PI-PLC同工酶的情况下为cIP和来自PI的肌醇-1-磷酸）。哺乳动物的酶，PI信号的关键调节器，在细胞生长和增殖中具有关键作用。分泌的细菌PI-PLC酶是相当小的单结构域蛋白，有助于宿主细胞的感染性。两种细菌（来自苏云金芽孢杆菌和单核细胞增生李斯特菌）的晶体结构，以及哺乳动物同工酶中最小的（85 kDa），PLCdelta 1识别出（β/α）8桶（哺乳动物酶的催化结构域）边缘暴露的疏水残基，这些残基可能与膜相互作用。我们试图表征这些界面残基激活和/或抑制酶活性的机制。诱变和多种生物物理学（例如，荧光、磁共振）方法将用于这些研究。 一个非常新颖的技术，高分辨率31 P场循环，将被用来表征的运动和构象的条件下，动力学激活的酶的底物。 这项工作的结果应该提供PI-PLC酶的（β/α）桶如何与膜表面相互作用的详细图片，包括蛋白质如何改变底物磷脂的动态特性，以及如何将该界面位点的结合转化为增强的催化作用。 鉴于哺乳动物PI-PLC在信号转导中的重要性，结果可能表明调节这些活动的新模式。以L.单核细胞增多症是一种人类细胞内的病原体，可以导致免疫功能低下的个体严重感染，这项工作也应该提供深入了解酶如何帮助细菌逃离吞噬体。

DESCRIPTION (provided by applicant): Phosphatidylinositol-specific phospholipase C enzymes catalyze the cleavage of phosphoinositides to diacylglycerol (a second messenger activator of protein kinase C), and inositol phosphates (cyclic 1,2-inositol phosphate in the case of bacterial PI-PLC and both cIP and inositol-1-phosphate from PI for mammalian Pl-PLC isozymes). The mammalian enzymes, key regulators of PI-signaling, have critical roles in cell growth and proliferation. The secreted bacterial PI-PLC enzymes, considerably smaller and single domain proteins, aid in infectivity of host cells. Crystal structures of two bacterial (from Bacillus thuringiensis and Listeria monocytogenes), and the smallest (85 kDa) of the mammalian isozymes, PLCdelta1 identify exposed hydrophobic residues at the rim of the (beta/alpha)8-barrel (the catalytic domain of the mammalian enzyme) that are likely to interact with membranes. We seek to characterize the mechanisms by which these interfacial residues activate and/or inhibit the enzyme activity. A combination of mutagenesis and diverse biophysical (e.g., fluorescence, magnetic resonance) methods will be used for these studies. A very novel technique, high resolution 31P field cycling, will be used to characterize the motion and conformation of substrates under conditions that kinetically activate the enzymes. The results from this work should provide a detailed picture of how the (beta/alpha)-barrel of PI-PLC enzymes interacts with the membrane surface, including how the protein alters dynamic characteristics of the substrate phospholipid, and how binding at this interfacial site is translated to enhanced catalysis. Given the importance of the mammalian PI-PLCs in signal transduction, the results may suggest new modes of modulating these activities. In the case of the PI-PLC from L. monocytogenes, an intracellular pathogen of humans that can cause serious infections in immunocompromised individuals, the work should also provide insight into how the enzyme aids in escape of the bacterium from the phagosome.