P4-ATPase mechanism of phospholipid translocation

P4-ATP酶磷脂易位机制

• 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
• 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

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)：细胞的定义特征是分隔细胞内外环境的令人难以置信的薄膜片。这种细胞膜，或称质膜，有一种脂类成分，可以阻挡极性或带电分子的通过，还有一种蛋白质成分，允许特权分子通过，使膜具有选择性的通透性。磷脂是质膜的关键组成部分，这些两亲性分子并排包装在一起，形成两层膜。每个小叶中的极性磷脂头基向外与水相互作用，疏水脂肪酰链面对双层结构的内部。真核细胞膜的一个显著特征是，这两层磷脂成分截然不同，这种现象被称为“膜不对称”。面向哺乳动物细胞胞浆的内叶富含磷脂酰丝氨酸(PS)和磷脂酰乙醇胺(PE)，而胞外小叶富含鞘脂、鞘糖脂和磷脂酰胆碱(PC)。至少有两类活性转运蛋白能够通过膜双层运动磷脂来建立膜的不对称性，包括IVP型ATPase(P4-ATPase)和ATP结合盒(ABC)转运体家族成员。令人惊讶的是，即使经过几十年的研究和一些ABC转运蛋白的X射线晶体结构，脂质是如何运输的仍然是一个谜。对于离子转运的P型ATPase(I、II和III型)，X射线晶体结构也得到了解决，突出了跨膜结构域中心的结构保守的底物结合部位(典型部位)。究竟是P4-ATPase进化出了识别这个典型位点上更大的磷脂底物的能力，还是进化出了一种独特的转运机制，一直是争论的主题。支持这一提议的初步数据有力地表明，P4-ATPase正在使用一种非规范的运输途径来翻转其磷脂底物。我们认为磷脂是在胞外小叶附近的“入口”和胞质小叶附近的“出口”处被选择的。该项目的研究将通过突变研究来验证这一双门假说，并更好地确定P4-ATPase转运磷脂的机制。所获得的信息将有助于我们理解人类疾病是如何由膜不对称缺陷或P4-ATPase缺陷引起的。