Somatic cell mutant affecting cholesterol transport

描述(申请人提供)：哺乳动物细胞严格控制它们的胆固醇含量和分布。在肝脏中，细胞胆固醇向内质网(ER)的运动是其转化为胆固醇酯并随后并入新生脂蛋白的关键。它也是肝脏胆固醇代谢为胆汁酸所必需的。胆固醇转运到内质网的途径和机制尚不清楚。阐明负责胆固醇向内质网运动的细胞因子可能会揭示高胆固醇血症的新治疗靶点。 我们已经分离到一个细胞膜胆固醇向ER(3，4)转运有缺陷的体细胞突变体。中国仓鼠卵巢(CHO)突变体3-6将新合成的胆固醇和脂蛋白衍生的胆固醇转运到质膜上，但不能将细胞表面的胆固醇动员到内质网进行代谢或调节稳态反应。这种表型可能是由于质膜脂质成分的变化，因为尽管3-6质膜中的胆固醇水平增加，3-6细胞对两性霉素B和在富含胆固醇的膜上形成毛孔的多烯抗生素具有抗药性。 我们的假设是，3-6基因编码一种影响质膜脂质组织的蛋白质。3-6活性的丧失改变了质膜脂域结构，使胆固醇对两性霉素B具有潜伏性，并阻止其进入其内吞途径。这项研究的目的是： 具体目标#1：鉴定当在突变的3-6细胞中表达时，纠正胆固醇运输缺陷表型的cDNA。表达亲生性逆转录病毒受体的3-6细胞将被导入逆转录病毒cDNA文库，将被鉴定为纠正表型的cDNA。我们将确定哪一个正确的cDNA编码3-6蛋白。 特定目的#2：鉴定其表达水平因3-6突变而改变的蛋白质。通过双向凝胶电泳法和微阵列分析对CHO和3-6蛋白质组和基因表达谱进行比较，揭示了3-6突变改变的候选3-6蛋白质和途径。来自这两种方法的候选人都将得到验证。具体目标#3：确定3-6诱导的细胞脂代谢变化。我们将确定3-6突变是如何改变质膜和/或内质网脂质成分的。鞘脂运输将在亲本CHO细胞和突变体3-6中进行检查。我们将研究候选3-6和抑制表型的蛋白质调节这些膜参数的机制。

DESCRIPTION (provided by applicant): Mammalian cells tightly regulate their cholesterol content and distribution. In the liver, the movement of cellular cholesterol to the endoplasmic reticulum (ER) is critical for its conversion to cholesteryl esters and subsequent incorporation into nascent lipoproteins. It is also essential for metabolism of hepatic cholesterol to bile acids. The route and mechanism of cholesterol transport to the ER is unknown. Elucidation of cellular factors responsible for cholesterol movement to the ER may reveal new therapeutic targets for hypercholesterolemia. We have isolated a somatic cell mutant with defective trafficking of plasma membrane cholesterol to the ER (3,4). Chinese hamster ovary (CHO) mutant 3-6 transports both newly synthesized and lipoprotein-derived cholesterol to the plasma membrane, but fails to mobilize cell surface cholesterol to the ER for metabolism or regulation of homeostatic responses. This phenotype is likely due to a change in the plasma membrane lipid composition since, despite increased levels of cholesterol in the 3-6 plasma membranes, 3-6 cells are resistant to amphotericin B, and an polyene antibiotic that forms pores in cholesterol-rich membranes. Our hypothesis is that the 3-6 gene encodes a protein that affects the lipid organization of the plasma membrane. Loss of 3-6 activity alters the plasma membrane lipid domain structure such that cholesterol is both latent to amphotericin B and prevented from entering its endocytic pathway. The Aims of this study are: Specific Aim #1: To identify cDNAs that, when expressed in mutant 3-6 cells, correct the cholesterol transport defective phenotype. 3-6 cells expressing an ecotropic retroviral receptor will be transfected with a retroviral cDNA library, cDNAs that correct the phenotype will be identified. We will determine which of the correcting cDNAs encode the 3-6 protein. Specific Aim #2: To identify proteins whose expression levels are altered by the 3-6 mutation. Comparison of CHO vs. 3-6 proteomes and gene expression patterns by two-dimensional gel electrophoresis and microarray analysis, respectively, has revealed candidate 3-6 proteins and pathways altered by the 3-6 mutation. Candidates from both approaches will be validated. Specific Aim #3: To define the 3-6 induced changes in cellular lipid metabolism. We will determine how the 3-6 mutation alters the plasma membrane and/or ER lipid compositions. Sphingolipid trafficking will be examined in parental CHO cells and mutant 3-6. We will investigate the mechanism by which candidate 3-6 and proteins that suppress the phenotype modulate these membrane parameters.