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酶催化磷脂酰肌醇裂解成二酰甘油(蛋白激酶C的第二信使激活剂)和肌醇磷酸盐(对于细菌的PI-PLC来说是环状1，2-肌醇磷酸，对于哺乳动物的PI-PLC同工酶是CIP和肌醇-1-磷酸)。哺乳动物酶是PI信号的关键调节者，在细胞的生长和增殖中起着关键作用。分泌的细菌PI-PLC酶是相当小的单一结构域蛋白，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性李斯特菌)的晶体结构，以及哺乳动物同工酶中最小的(85 KDa)，PLCdelta1识别(β/α)8桶(哺乳动物酶的催化区域)边缘暴露的疏水残基，这些残基可能与膜相互作用。我们试图描述这些界面残基激活和/或抑制酶活性的机制。这些研究将结合使用诱变和不同的生物物理(如荧光、磁共振)方法。一种非常新的技术，高分辨率31p场循环，将被用来表征在动力学激活酶的条件下底物的运动和构象。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景，包括蛋白质如何改变底物磷脂的动态特性，以及该界面位置的结合如何转化为增强催化。鉴于哺乳动物PI-PLC在信号转导中的重要性，这一结果可能会提出调节这些活动的新模式。在单核细胞增多性李氏杆菌的PI-PLC的案例中，这项工作还应该提供对这种酶如何帮助细菌从吞噬小体中逃逸的洞察。单核细胞增多性李氏杆菌是人类的一种细胞内病原体，可导致免疫受损的个体发生严重感染。

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.