Activation of PI Specific Phospholipase C Enzymes

哺乳动物磷脂酰肌醇特异性磷脂酶C （PI-PLC）酶是多结构域蛋白，是磷脂酰肌醇信号传导的关键调节因子，在细胞生长和增殖中起关键作用。细菌PI-PLC酶是一种结构相当于哺乳动物酶的催化结构域的单域蛋白，通常分泌并有助于靶细胞的感染。虽然我们研究的三种蛋白苏云金芽孢杆菌PI-PLC、单核增生李斯特菌PI-PLC和哺乳动物PLC41具有共同的催化结构域结构框架，但它们与脂质的相互作用和调节是不同的，并且与它们各自的生理功能密切相关。我们的长期目标是得出这些蛋白质如何停靠在目标双层膜上的分子水平图像。具体来说，我们计划(i)使用高分辨率场循环31P核磁共振弛豫实验来定义和表征特定的非底物/变构磷脂结合位点；（ii）使用NMR和HXMS方法将这些pi - plc定位在模型膜上，特别是关于活性位点周围的移动环和疏水脊残基；（iii）使用FCS和单分子荧光显微镜将PI-PLC结合研究从简单的囊泡扩展到更复杂的靶细胞样膜。这项工作的结果应该显示这些酶如何与膜表面的不同组分相互作用，以及如何在非底物界面位点结合转化为增强的催化作用。单核增生乳杆菌是一种人类细胞内病原体，可引起免疫功能低下个体的严重感染。深入了解这种有助于毒力的PI-PLC如何与胞内膜相互作用，可能提供减缓这种生物体胞内生长的替代方法。同样，一些蜡样芽孢杆菌菌株是机会性病原体，PI-PLC对gpi锚定蛋白的裂解可能有助于毒性。对于PLC41，重点是通过无序肽区域对酶的自抑制，这可能是控制酶基础活性的重要途径。

DESCRIPTION (provided by applicant): Mammalian phosphatidylinositol specific phospholipase C (PI-PLC) enzymes are multidomain proteins that are key regulators of phosphoinositide-signaling, They have critical roles in cell growth and proliferation. The bacterial PI-PLC enzymes, single domain proteins structurally equivalent to the catalytic domain of the mammalian enzymes, are usually secreted and often aid in the infection of target cells. Although the three proteins we study, Bacillus thuringiensis PI-PLC, Listeria monocytogenes PI-PLC, and mammalian PLC41, share a common structural framework for the catalytic domain, their interaction with and regulation by lipids is different and tied up with their individual physiological functions. Our long-term goal is to derive a molecular level picture of how these proteins dock on their target bilayer membranes. Specifically, we plan to (i) define and characterize specific nonsubstrate/allosteric phospholipid binding site(s) using high resolution field cycling 31P NMR relaxation experiments; (ii) orient these PI-PLCs on model membranes, particularly with respect to the mobile loops and hydrophobic ridge residues surrounding the active sites using NMR and HXMS methods; and (iii) extend PI-PLC binding studies, using FCS and single molecule fluorescence microscopy, from simple vesicles to more complex target cell-like membranes. The results from this work should show how these enzymes interact with different components of the membrane surface and how binding at nonsubstrate interfacial sites is translated to enhanced catalysis. L. monocytogenes is an intracellular pathogen of humans that can cause serious infections in immunocompromised individuals. Insight into how this PI-PLC, which contributes to virulence, interacts with intracellular membranes may provide alternate ways of slowing intracellular growth of this organism. Likewise some Bacillus cereus strains are opportunistic pathogens and PI-PLC cleavage of GPI-anchored proteins is likely to contribute to virulence. For PLC41, the emphasis is on autoinhibition of the enzyme by a disordered peptide region that may be an important way of controlling basal activity of the enzyme. PUBLIC HEALTH RELEVANCE: This research will provide new insights into how phospholipids in membranes interact with and modulate the activities of enzymes that transiently bind to the membrane. The enzymes examined are a class of phospholipases that catalyze the same chemistry but have different physiological roles - one is mammalian and critical to cell growth and proliferation, while two are bacterial and involved in infectivity of the organisms. Characterizing the membrane interactions of the mammalian enzyme contributes to understanding what happens in cells in a variety of diseases where cell proliferation is unchecked. The studies of the bacterial enzymes may provide new treatments for slowing/inhibiting infections.