Activation of PI Specific Phospholipase C Enzymes

描述（由申请方提供）：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，是磷酸肌醇信号传导的关键调节因子，在细胞生长和增殖中发挥关键作用。细菌PI-PLC酶是结构上等同于哺乳动物酶的催化结构域的单结构域蛋白，通常是分泌的，并且通常有助于感染靶细胞。虽然我们研究的三种蛋白质，苏云金芽孢杆菌PI-PLC，单核细胞增生李斯特菌PI-PLC和哺乳动物PLC 41，共享一个共同的结构框架的催化结构域，它们的相互作用和调节脂质是不同的，并捆绑在一起与他们各自的生理功能。我们的长期目标是从分子水平上了解这些蛋白质是如何停靠在其目标双层膜上的。具体而言，我们计划（i）使用高分辨率场循环31 P NMR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方法来在模型膜上定位这些PI-PLC，特别是关于活性位点周围的移动的环和疏水脊残基;和（iii）扩展PI-PLC结合研究，使用FCS和单分子荧光显微镜，从简单的囊泡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。L.单核细胞增多症是人类的细胞内病原体，其可在免疫功能低下的个体中引起严重感染。深入了解这种PI-PLC如何与细胞内膜相互作用，这有助于毒力，可能会提供减缓这种生物体细胞内生长的替代方法。同样，一些蜡样芽孢杆菌菌株是机会致病菌，GPI锚定蛋白的PI-PLC裂解可能有助于毒力。对于PLC 41，重点是通过无序肽区域对酶的自抑制，这可能是控制酶的基础活性的重要方式。 公共卫生相关性：这项研究将提供新的见解，如何磷脂在膜相互作用，并调节酶的活动，瞬时结合到膜。所研究的酶是一类磷脂酶，其催化相同的化学反应，但具有不同的生理作用-一种是哺乳动物的，对细胞生长和增殖至关重要，而两种是细菌的，涉及生物体的感染性。表征哺乳动物酶的膜相互作用有助于理解在细胞增殖未受抑制的各种疾病中细胞中发生的情况。对细菌酶的研究可能为减缓/抑制感染提供新的治疗方法。

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.