Molecular mechanism of phospholipid scrambling by rhodopsin

视紫红质是一种具有结构性活性的脂类扰乱酶，能够移动磷脂。 迅速地在膜双层的小叶之间。视紫红质的这种新的活性在 使ABC转运蛋白ABCA4能够防止视黄醛的积累并减轻形成 维生素A二聚体。这些分子在视网膜中的有毒积聚被认为是导致 视网膜病变的潜在机制，如Stargardt病和老年性黄斑变性。 视紫红质及其脱辅蛋白视蛋白都是扰乱酶，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类在特定分子的界面上运输 视蛋白中的跨膜螺旋。质膜缺乏结构性磷脂扰乱酶活性，并且 从而将信号脂质磷脂酰丝氨酸(PS)隔离在内叶中，直到扰乱酶 激活了。然而，过度表达视蛋白的细胞出人意料地没有在其表面显示PS。我们 假设质膜中的高胆固醇水平抑制了视蛋白的扰乱酶活性。我们 提出两个具体的目标，通过生物物理、生物化学和计算方法来验证这些假说。 在第一个目标中，我们将结合实验和电子分析来测试脂转移途径。 在我们的分子动力学模拟预测的视蛋白内。使用定点突变，我们将 交联性跨膜螺旋，并改变途径环境，以扰乱运输，AS 在我们的基于荧光的扰乱酶检测中进行了测量。我们还将对突变的opsins进行分子分析 动力学模拟，以获得结构背景和对实验的力学见解 结果。在第二个目标中，我们将使用基于细胞和囊泡的分析来确定胆固醇对 脂质扰乱，结合胆固醇中野生型视蛋白的分子动力学模拟- 含有膜的。 拟议中的研究将首次提供对脂质扰乱机制的见解。作为加扰器 它们都是最近才被发现的，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未来详细阐明这一过程奠定基础。 这对生理学有重要的意义，因为视蛋白的扰乱机制是 可能在支持光感受器盘膜ABCA4的翻转酶活性方面有作用。的不足之处 这种机制显然与视网膜病变有关。此外，因为视蛋白的扰乱酶活性是 我们的实验与其他G蛋白偶联受体(GPCR)共享，如β2-肾上腺素能受体 也有可能开辟GPCR生物学的一个新领域。

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.