P4-ATPase mechanism of phospholipid translocation

P4-ATP酶磷脂易位机制

基本信息

批准号：
8724534
负责人：
TODD R GRAHAM
金额：
$ 29.82万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8724534
关键词：
ATP Hydrolysis ATP phosphohydrolase ATP-Binding Cassette Transporters Active Biological Transport Amino Acids Apoptotic Binding Binding Sites Biochemical Blood coagulation Carrier Proteins Cations Cell Polarity Cell membrane Cells Characteristics Charge Chimera organism Cleaved cell Coupling Crosslinker Cysteine Cytokinesis Cytosol Data Defect Disease Environment Eukaryota Eukaryotic Cell Excision Face Family Glycosphingolipids Golgi Apparatus H(+)-K(+)-Exchanging ATPase Homology Modeling Human Immune Intrahepatic Cholestasis Ion Transport Learning Lecithin Lipids Male Infertility Malignant neoplasm of liver Mammalian Cell Mammals Maps Mediating Membrane Membrane Proteins Mental Retardation Modeling Molecular Genetics Mus Mutagenesis Mutation Na(+)-K(+)-Exchanging ATPase Non-Insulin-Dependent Diabetes Mellitus Obesity Pathway interactions Peripheral Phenylalanine Phosphatidylethanolamine Phosphatidylserines Phospholipids Positioning Attribute Progressive intrahepatic cholestasis Proteins Pump Resolution Role Saccharomycetales Series Side Signal Transduction Site Sphingolipids Structural Models Structure Subgroup Substrate Specificity System Testing Transmembrane Domain Vesicle Viral Water base crosslink extracellular hearing impairment human disease humoral immunity deficiency insight lipid transport member novel protein transport public health relevance screening

项目摘要

DESCRIPTION (provided by applicant): The defining feature of a cell is the incredibly thin sheet of membrane that demarcates the intracellular and extracellular milieus. This cell membrane, or plasma membrane, has a lipid component that provides a barrier to passage of polar or charged molecules, and protein component that allows passage of privileged molecules to make the membrane selectively permeable. Phospholipids are critical building blocks of the plasma membrane and these amphipathic molecules pack together side-by-side to form a two-layered sheet. The polar phospholipid headgroups in each leaflet face outward to interact with water and the hydrophobic fatty acyl chains face the interior of the bilayer structure. A remarkable characteristic of the eukaryotic cell membrane is that these two layers have a very different phospholipid composition, a phenomenon known as "membrane asymmetry". The inner leaflet facing the cytosol of mammalian cells is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) while the extracellular leaflet is enriched in sphingolipids, glycosphingolipids and phosphatidylcholine (PC). At least two classes of active-transport proteins are capable of moving phospholipids across a membrane bilayer to establish membrane asymmetry, including the type IV P-type ATPases (P4-ATPases) and members of the ATP-binding cassette (ABC) transporter families. Amazingly, even with decades of study and x-ray crystal structures of some ABC transporters, how lipids are transported remains a mystery. With ion- transporting P-type ATPases (type I, II and III), x-ray crystal structure have also been solved highlighting a structurally conserved substrate binding site in the center of the transmembrane domain (a canonical site). Whether P4-ATPases evolved the ability to recognize the much larger phospholipid substrate in this canonical site, or evolved a unique transport mechanism has been the subject of debate. Preliminary data supporting this proposal strongly suggests that the P4-ATPases are using a noncanonical transport pathway to flip their phospholipid substrate. We propose that phospholipid is being selected at both an "entry gate" near the extracellular leaflet and an "exit gate" near the cytosolic leaflet. Studies in this projet will test this two-gate hypothesis through mutational studies and better define the mechanism of phospholipid transport by the P4- ATPases. The information obtained will help us understand how human diseases arise from defects in membrane asymmetry or P4-ATPase deficiency.

描述（由申请人提供）：细胞的定义特征是极其薄的膜，它划分细胞内和细胞外环境。这种细胞膜或质膜具有脂质成分和蛋白质成分，脂质成分为极性或带电分子的通过提供屏障，蛋白质成分允许特权分子通过以使膜选择性渗透。磷脂是质膜的关键组成部分，这些两亲性分子并排堆积在一起形成双层片。每个小叶中的极性磷脂头基面向外与水相互作用，疏水性脂肪酰基链面向双层结构的内部。真核细胞膜的一个显着特征是这两层具有非常不同的磷脂组成，这种现象被称为“膜不对称”。面向哺乳动物细胞胞浆的内部小叶富含磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE），而细胞外小叶富含鞘脂、糖鞘脂和磷脂酰胆碱（PC）。至少两类主动转运蛋白能够使磷脂穿过膜双层以建立膜不对称性，包括 IV 型 P 型 ATP 酶 (P4-ATP 酶) 和 ATP 结合盒 (ABC) 转运蛋白家族的成员。令人惊讶的是，即使经过数十年的研究和一些 ABC 转运蛋白的 X 射线晶体结构，脂质如何转运仍然是一个谜。对于离子传输 P 型 ATP 酶（I 型、II 型和 III 型），X 射线晶体结构也已得到解决，突出显示跨膜结构域中心的结构保守的底物结合位点（典型位点）。 P4-ATP酶是否进化出识别该经典位点中更大的磷脂底物的能力，或者进化出独特的转运机制一直是争论的主题。支持这一提议的初步数据强烈表明 P4-ATP 酶正在使用非规范运输途径来翻转其磷脂底物。我们建议磷脂在细胞外小叶附近的“入口门”和细胞质小叶附近的“出口门”处被选择。该项目的研究将通过突变研究检验这种双门假说，并更好地定义 P4-ATP 酶转运磷脂的机制。获得的信息将帮助我们了解人类疾病是如何由膜不对称缺陷或 P4-ATP 酶缺乏引起的。