Activation of PI Specific Phospholipase C Enzymes

描述(申请人提供)：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，是磷脂酰肌醇信号转导的关键调节因子，它们在细胞生长和增殖中起关键作用。细菌PI-PLC酶是一种结构上与哺乳动物酶的催化结构域相当的单域蛋白，通常是分泌的，通常有助于靶细胞的感染。尽管我们研究的三种蛋白质，苏云金芽孢杆菌PI-PLC、单核细胞增多性李斯特菌PI-PLC和哺乳动物PLC41具有共同的催化域结构框架，但它们与脂类的相互作用和调节是不同的，并且与各自的生理功能密切相关。我们的长期目标是得到这些蛋白质如何对接在它们的目标双层膜上的分子水平的图像。具体地说，我们计划(I)通过高分辨率场循环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(S)；(Ii)利用核磁共振和HXMS方法将这些PI-PLC定向到模型膜上，特别是关于活性中心周围的可移动环和疏水脊残基；以及(Iii)利用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成分相互作用，以及非底物界面位置的结合如何转化为增强催化。单核细胞增多性乳杆菌是人类的一种细胞内病原体，可在免疫功能低下的人中引起严重感染。深入了解这种对毒力有贡献的PI-PLC是如何与细胞内膜相互作用的，可能会提供减缓这种生物细胞内生长的替代方法。同样，一些蜡状芽孢杆菌菌株是条件致病菌，PI-PLC裂解GPI锚定蛋白可能有助于毒力。对于PLC41，重点是无序多肽区对酶的自动抑制，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.