Molecular Characterization of the Phospholipid Flippase Substrate Pathway

 描述(申请人提供)：在质膜内，双层的细胞面和腔/外表面的小叶由不同的磷脂种类组成。这种磷脂在双层内的不均匀分布称为膜磷脂不对称。这种不对称性协调了许多细胞功能，从膜弯曲到分泌功能，以及细胞内和细胞间信号传递。磷脂不对称的破坏与神经功能障碍、血液紊乱、胆汁淤积和2型糖尿病有关。然而，细胞维持质膜和内部细胞器的磷脂不对称性的方式还知之甚少。膜磷脂不对称的一个关键调节因素是一种称为磷脂翻转酶或P4型ATPase的酶家族。我的研究计划将使用正向遗传策略和定向酶进化来：i)确定用于区分磷脂骨架的P4-ATPase残基，ii)确定磷脂酰乙醇胺头基识别的分子基础，iii)测试负责底物保护和通过膜的初级结构机制。阐明这些机制对于了解细胞如何设置、维持、破坏和修复其膜的磷脂不对称至关重要。这些分子发现将对设计新的酶技术以干扰和检查神经系统、淋巴细胞、血管系统和肝脏系统中的膜不对称效应器至关重要。最后，我们预计，对底物识别和协调的分子理解将促进用于疾病治疗的药物疗法的生产。

 DESCRIPTION (provided by applicant): Within the plasma membrane, the cytofacial and lumenal/exofacial leaflets of the bilayer are known to be composed of different phospholipid species. This unequal distribution of phospholipids within the bilayer is referred to as membrane phospholipid asymmetry. This asymmetry is coordinates a number of cellular functions ranging from membrane curvature, to secretory function, and intra- and intercellular signaling. Disruption of phospholipid asymmetry has been linked to neurological dysfunction, blood disorders, cholestasis, and type 2 diabetes. However, the means through which a cell maintains phospholipid asymmetry of the plasma membrane and internal organelles is poorly understood. One critical regulator of membrane phospholipid asymmetry is a family of enzymes called phospholipid flippases, or P4-type ATPases. My research plan will use forward genetic strategies and directed enzyme evolution to: i) determine the P4- ATPase residues used to discriminate phospholipid backbone, ii) define the molecular basis for phosphatidylethanolamine headgroup recognition, and iii) test the primary structural mechanism responsible for substrate protection and passage through the membrane. Elucidating these mechanisms will be critical to understanding how the cell sets, maintains, disrupts, and repairs phospholipid asymmetry of its membranes. These molecular findings will be critical for the design of new enzyme technologies for the perturbation and examination membrane asymmetry effectors within the nervous system, lymphocytes, vasculature, and hepatic system. Finally, we anticipate that a molecular understanding of substrate recognition and coordination will facilitate the production of pharmaceutical therapies for disease treatment.