ASYMMETRIC DISTRIBUTION OF CHOLESTEROL IN MEMBRANES

胆固醇在细胞和细胞膜中的分布并不均匀。不是 到目前为止，单一的机制解释了细胞内胆固醇的运输。 固醇载体蛋白-2(SCP-2)介导的转导最少 明白了。SCP-2作为一个蛋白质家族存在[主要是13(SCP-2)，15 (Pro-SCP-2)、29(SCP-y)和58(SCP-x)kDa形式]来自单个 基因，共用一个13 kDa的C末端。它们在N端的不同之处 靶向序列、细胞内定位和推测的功能。 然而，关于是否有SCP-2甚至 结合胆固醇或关于这些普遍存在的蛋白质的机制(S) 在体外介导甾醇转移。除了一个例外，没有 有证据表明任何SCP-2在胆固醇运输中起作用 完好无损的细胞。在本应用中，重组SCP-2将用于 解决以下问题： 1)鉴定重组SCP-2的配体结合部位(S)。虽然 不存在与13 kDa SCP-2结合的内源性配体，荧光和 放射性标记的甾醇显示存在一个单一的甾醇结合部位。 这些研究将扩展到探索配体结合部位(S)。 15、29和58 kDa的SCP-2。 2)研究SCP-2形式在靶向胆固醇中的作用 胞内膜组分与细胞器之间的体外转移。 13 kDa的SCP-2差异转运固醇：质膜和GT； 微体&线粒体。由所有SCP-2表格针对特定 膜将被确定。 3)检测SCP-2形式在细胞内的分布和作用 完整细胞内的类固醇转运和外流。已转染 表达13或15 kDa SCP-2s的L细胞增强摄取和酯化 中间体源性胆固醇和质膜氧化的关系 胆固醇。四种SCP-2在胆固醇外流和细胞凋亡中的作用 将在转基因细胞中检测细胞内的转运。 4)体外研究SCP-2的形成调控机制 膜内胆固醇结构域。13 kDa的SCP-2重新分布 胆固醇在体外模型膜和质膜中的作用 加强类固醇的解吸。四种SCP-2对细胞膜的影响 将在转基因细胞中确定甾醇结构域。 这些实验应该会对细胞的功能产生新的见解 SCP-2s，为胆固醇结构域的调控提供了新的数据。 这将有助于我们理解涉及异常的疾病 胆固醇的吸收以及胆固醇的运输和/或 积累(过氧化物酶缺乏症、癌症、动脉粥样硬化、衰老)。

Cholesterol is not uniformly distributed in the cell and in membranes. No single mechanism to date explains intracellular cholesterol trafficking. Sterol carrier protein-2 (SCP-2) mediated transfer is the least understood. SCP-2 exists as a family of proteins [primarily 13 (SCP-2), 15 (pro-SCP-2), 29 (SCP-y), and 58 (SCP-x) kDa forms] arising from a single gene and sharing a common 13 kDa C-terminus. They differ in N-terminal targeting sequences, intracellular localization, and presumably function. However, there is as yet no general agreement on whether any SCP-2s even bind cholesterol or on the mechanism(s) whereby these ubiquitous proteins mediate sterol transfer in vitro. With a single exception there is no evidence that any of the SCP-2s function in cholesterol trafficking in intact cells. In this application recombinant SCP-2s will be used to address these issues: 1) Characterize the ligand binding site(s) of recombinant SCP-2s. Although no endogenous ligand copurified with 13 kDa SCP-2, fluorescent and radiolabeled sterols show the presence of a single sterol binding site. These studies will be extended to explore the ligand binding site(s) of the 15, 29, and 58 kDa SCP-2s. 2) Investigate the role of the SCP-2 forms in targeting cholesterol transfer between intracellular membrane fractions and organelles in vitro. The 13 kDa SCP-2 differentially transfers sterol: plasma membranes > microsomes >> mitochondria. Targeting by all SCP-2 forms to specific membranes will be determined. 3) Examine the intracellular distribution and role of the SCP-2 forms on intracellular sterol trafficking and efflux in intact cells. Transfected L-cells expressing 13 or 15 kDa SCP-2s enhanced uptake and esterification of medium derived cholesterol and oxidation of plasma membrane derived cholesterol. The role of all four SCP-2s in cholesterol efflux and intracellular trafficking will be examined in transfected cells. 4) Explore in vitro the mechanism whereby the SCP-2 forms regulate intramembrane cholesterol domain structure. The 13 kDa SCP-2 redistributes cholesterol within model membranes and plasma membranes in vitro to enhance sterol desorption. The effect of all four SCP-2s on membrane sterol domains will be determined in transfected cells. These experiments should yield novel insights into the function of the SCP-2s and provide new data on regulation of cholesterol domain structure. This will contribute to our understanding of diseases involving abnormal cholesterol absorption as well as cholesterol trafficking and/or accumulation (peroxisomal deficiency, cancer, atherosclerosis, aging).