ASYMMETRIC DISTRIBUTION OF CHOLESTEROL IN MEMBRANES

胆固醇在细胞和细胞膜中的分布并不均匀。没有 迄今为止，只有一种机制可以解释细胞内胆固醇的运输。 甾醇载体蛋白-2（SCP-2）介导的转移最少 明白SCP-2作为蛋白质家族存在[主要是13（SCP-2），15 （pro-SCP-2），29（SCP-y）和58（SCP-x）kDa形式]产生于单个 基因和共享一个共同的13 kDa的C-末端。它们在N-末端 靶向序列、细胞内定位和推测的功能。 然而，目前还没有普遍的共识，是否有任何SCP-2甚至 结合胆固醇或这些无处不在的蛋白质 介导甾醇转移。除了一个例外， 有证据表明任何SCP-2的功能都是在胆固醇运输中， 完整的细胞在本申请中，重组SCP-2将用于 解决这些问题： 1)表征重组SCP-2的配体结合位点。虽然 无内源性配体与13 kDa SCP-2共纯化，荧光和 放射性标记的甾醇显示存在单个甾醇结合位点。 这些研究将扩展到探索配体结合位点（S） 15、29和58 kDa SCP-2。 2)研究SCP-2形式在靶向胆固醇中的作用 在体外细胞内膜组分和细胞器之间的转移。 13 kDa SCP-2差异转移固醇：质膜> 微粒体>>线粒体。所有SCP-2形式的目标是特定的 膜将被确定。 3)检查SCP-2形式在细胞内的分布和作用， 完整细胞中的细胞内固醇运输和流出。转染 表达13或15 kDa SCP-2s的L-细胞增强摄取和酯化 培养基来源的胆固醇和质膜来源的氧化 胆固醇所有四个SCP-2在胆固醇流出和 将在转染细胞中检查细胞内运输。 4)体外探索SCP-2形式调节的机制 膜内胆固醇结构域。13 kDa SCP-2重新分配 胆固醇在体外模型膜和质膜内， 增强甾醇解吸。四种SCP-2对细胞膜的影响 在转染的细胞中测定甾醇结构域。 这些实验应该产生新的见解的功能， SCP-2s，并提供了关于胆固醇结构域调节的新数据。 这将有助于我们了解涉及异常的疾病 胆固醇吸收以及胆固醇运输和/或 积累（过氧化物酶体缺乏，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).