Molecular mechanism of phospholipid scrambling by rhodopsin

视紫红质扰乱磷脂的分子机制

• 批准号: 9374546
• 负责人: George Khelashvili
• 金额: $ 25.43万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9374546
• 关键词: ATP-Binding Cassette Transporters; Adolescent; Affect; Age related macular degeneration; Amino Acids; Animals; Apoproteins; Area; Biochemical; Biological Assay; Biology; Biophysics; Cell membrane; Cells; Cholesterol; Computer Simulation; Computing Methodologies; Cyclodextrins; Data; Dimerization; Disease; Environment; Fluorescence; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Goals; Hydration status; Knowledge; Lead; Light; Lipids; Maps; Measures; Mediating; Membrane; Molecular; Opsin; Pathway interactions; Phosphatidylserines; Phospholipids; Photoreceptors; Physiology; Play; Population; Process; Proteins; Reader; Retina; Retinal; Retinal Diseases; Retinal Pigments; Retinaldehyde; Retinoids; Rhodopsin; Role; Route; Side; Signal Transduction; Site-Directed Mutagenesis; Stargardt' s disease; Surface; Techniques; Testing; Transmembrane Transport; Transport Process; Vesicle; Vestibule; Vitamin A; Water; Work; base; beta-2 Adrenergic Receptors; computer studies; conformational conversion; crosslink; dimer; disulfide bond; experimental study; infancy; insight; interest; lipid transport; molecular dynamics; mutant; novel; overexpression; phospholipid scramblase; photoreceptor disc; prevent; reconstitution; unilamellar vesicle

The visual pigment rhodopsin is a constitutively active lipid scramblase capable of moving phospholipids rapidly between the leaflets of a membrane bilayer. This novel activity of rhodopsin plays a key role in enabling the ABC transporter ABCA4 to prevent accumulation of retinaldehydes and mitigate the formation of Vitamin A dimers. The toxic buildup of these molecules in the retina is considered to contribute to mechanisms underlying retinopathies such as Stargardt's disease and age-related macular degeneration. Both rhodopsin and its apo-protein opsin are scramblases, and here we are interested in understanding the molecular mechanism by which they translocate phospholipids. Based on preliminary results from atomistic molecular dynamics simulations we hypothesize that lipids are transported at the interface of specific transmembrane helices in opsin. Plasma membranes lack constitutive phospholipid scramblase activity, and thereby sequester the signaling lipid phosphatidylserine (PS) in the inner leaflet until scramblases are activated. However, cells that over-express opsin unexpectedly do not display PS at their surface. We hypothesize that high cholesterol levels in the plasma membrane silence opsin's scramblase activity. We propose two specific aims to test these hypotheses via biophysical, biochemical and computational methods. In the first aim we will combine experimental and in silico analyses to test the lipid translocation pathway within opsin predicted by our molecular dynamics simulations. Using site-directed mutagenesis we will crosslink transmembrane helices and also alter the pathway environment in order to disrupt transport, as measured in our fluorescence-based scramblase assays. We will also analyze the mutant opsins by molecular dynamics simulations in order to obtain structural context and mechanistic insights into the experimental results. In the second aim, we will use cell and vesicle-based assays to determine the effect of cholesterol on lipid scrambling, in conjunction with molecular dynamics simulations of wild-type opsin in cholesterol- containing membranes. The proposed studies will provide the first insights into the mechanism of lipid scrambling. As scramblases have only recently been discovered, their molecular mechanism is unknown and the field is in its infancy. The knowledge that we gain here will set the stage for future elucidation of this process in molecular detail. This has important implications for physiology, because of the role that opsin's scramblase mechanism is likely to have in supporting the flippase activity of ABCA4 in photoreceptor disc membranes. Deficiencies in this mechanism are clearly associated with retinopathies. Moreover, because opsin's scramblase activity is shared by other G protein-coupled receptors (GPCRs) such as the β2-adrenergic receptor, our experiments also have the potential to open a new area of GPCR biology.

视色素视紫红质是一种组成型活性脂质扰乱酶，能够移动磷脂 快速地在膜双层的小叶之间。视紫红质的这种新颖活性在 使 ABC 转运蛋白 ABCA4 能够防止视黄醛的积累并减少其形成 维生素A二聚体。这些分子在视网膜中的毒性积累被认为有助于 斯塔加特氏病和年龄相关性黄斑变性等视网膜病变的潜在机制。 视紫红质及其脱辅基蛋白视蛋白都是乱序酶，在这里我们有兴趣了解 它们转运磷脂的分子机制。基于原子论的初步结果 分子动力学模拟我们假设脂质在特定的界面上运输 视蛋白中的跨膜螺旋。质膜缺乏组成型磷脂扰乱酶活性，并且 从而将信号脂质磷脂酰丝氨酸 (PS) 隔离在内部小叶中，直到乱序被 活性。然而，过度表达视蛋白的细胞出乎意料地不会在其表面显示 PS。我们 假设质膜中的高胆固醇水平会抑制视蛋白的扰乱酶活性。我们 提出了两个具体目标，通过生物物理、生物化学和计算方法来检验这些假设。 在第一个目标中，我们将结合实验和计算机分析来测试脂质易位途径 在我们的分子动力学模拟预测的视蛋白内。使用定点诱变，我们将 交联跨膜螺旋并改变通路环境以破坏运输，如 在我们基于荧光的扰乱酶测定中进行测量。我们还将通过分子分析分析突变视蛋白 动力学模拟以获得实验的结构背景和机制见解 结果。在第二个目标中，我们将使用基于细胞和囊泡的检测来确定胆固醇对 脂质扰乱，结合胆固醇中野生型视蛋白的分子动力学模拟 含有膜。 拟议的研究将为脂质扰乱机制提供初步见解。作为乱序 最近才被发现，其分子机制尚不清楚，该领域还处于起步阶段。 我们在这里获得的知识将为将来阐明这一过程的分子细节奠定基础。 这对生理学具有重要意义，因为视蛋白的扰乱酶机制在 可能支持感光盘膜中 ABCA4 的翻转酶活性。不足之处 这种机制显然与视网膜病变有关。此外，由于视蛋白的扰乱酶活性 与其他 G 蛋白偶联受体 (GPCR) 共享，例如 β2-肾上腺素受体，我们的实验 也有可能开辟 GPCR 生物学的新领域。