ETHANOL EFFECT ON MEMBRANE PROTEIN/LIPID INTERACTIONS

乙醇对膜蛋白/脂质相互作用的影响

• 批准号: 6012286
• 负责人: CHRISTOPHER D STUBBS
• 金额: $ 28.59万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6012286
• 关键词: Baculoviridae; G protein; Insecta; alcoholic beverage consumption; animal tissue; beta adrenergic receptor; cell line; chemical hydration; complementary DNA; conformation; ethanol; hydrogen bond; hydropathy; membrane activity; membrane lipids; membrane proteins; molecular site; phospholipids; point mutation; protein protein interaction; protein purification; protein reconstitution; protein structure function; receptor coupling; tissue /cell culture; transfection; vasopressins

It is proposed to investigate the hypothesis that ethanol resides at sites on the hydrophobic surface and in inter-helical regions of trans-membrane proteins where it modifies the ability of the protein to change its conformation as part of normal functioning. It is proposed that "sites" of interaction may be quite common, although few are likely to provide a significant enough interaction to modulate function. The ethanol interaction partly involves hydrophobic interactions but in addition a weakening of a protein-lipid hydrogen bonding network that include water molecules at sensitive regions on the protein is proposed to play a major role. Published work from this laboratory on lipid bilayer membranes with model membrane proteins inserted indicate that ethanol significantly perturbs the phospholipid head group, acyl chain and protein-lipid interfacial regions in membranes by weakening the hydrogen bond network. In addition evidence that chronic-ethanol ingestion leads to adaptive changes in the cell membrane lipids that attenuate this (direct) effect of ethanol has been presented. In this renewal it proposed to test the hypothesis of a link between an ethanol-induced perturbation of the hydrogen bond network and effects on function in seven-transmembrane G-protein coupled receptors (GPCR) which are a large family of proteins that initiate numerous signal transduction cascades in cells. The cDNA for two representative GPCR: the vasopressin-V1A and beta2-adrenergic receptors has been inserted into baculovirus vectors and expressed in Sf9 insect cells and will be affinity purified and reconstituted into defined lipids. The reconstitution will include lipids possessing the chronic ethanol-induced modification, to test the role of perturbation the hydrogen bond network on the agonist- induced trans-membrane inter- and intra-helical conformational changes that leads to G-protein activation. Putative regions that might harbor ethanol "sites" and that would be likely to lead to functional perturbation will be further delineated using point mutations.

建议研究以下假设：乙醇驻留在跨膜蛋白的疏水表面和螺旋间区域的位点，在那里它改变蛋白质改变其构象的能力，作为正常功能的一部分。有人提出，相互作用的“位点”可能相当常见，尽管很少有可能提供足够重要的相互作用来调节功能。 乙醇相互作用部分涉及疏水相互作用，但此外，蛋白质-脂质氢键网络的减弱（包括蛋白质敏感区域的水分子）被认为发挥了主要作用。 该实验室发表的关于插入模型膜蛋白的脂质双层膜的研究表明，乙醇通过削弱氢键网络显着扰乱膜中的磷脂头基、酰基链和蛋白质-脂质界面区域。 此外，有证据表明，长期摄入乙醇会导致细胞膜脂质发生适应性变化，从而减弱乙醇的这种（直接）作用。 在这次更新中，它提出测试乙醇诱导的氢键网络扰动与七跨膜 G 蛋白偶联受体 (GPCR) 功能影响之间存在联系的假设，GPCR 是一个启动大量信号的蛋白质大家族细胞内的转导级联。 两个代表性 GPCR：加压素-V1A 和 β2-肾上腺素受体的 cDNA 已插入杆状病毒载体中并在 Sf9 昆虫细胞中表达，并将进行亲和纯化并重构为确定的脂质。 重构将包括具有慢性乙醇诱导修饰的脂质，以测试氢键网络扰动对激动剂诱导的跨膜螺旋间和螺旋内构象变化（导致 G 蛋白激活）的作用。 将使用点突变进一步描绘可能含有乙醇“位点”并且可能导致功能扰动的假定区域。