Regulation of stearoyl-CoA desaturases by oleate

油酸对硬脂酰辅酶A去饱和酶的调节

• 批准号: 6613483
• 负责人: DOUGLAS P THEWKE
• 金额: $ 17.32万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613483
• 关键词: CHO cells; DNA footprinting; acetyl coA; cell proliferation; cholesterol; enzyme activity; fatty acid biosynthesis; gel mobility shift assay; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; membrane reconstitution /synthesis; oleate; oxidoreductase; polymerase chain reaction; protein binding; regulatory gene; steroid biosynthesis; tissue /cell culture; western blottings

DESCRIPTION: (Scanned from the applicant's abstract) The long term objective is to understand the mechanisms by which C18 fatty acids regulate the synthesis of cholesterol and fatty acids necessary for the synthesis of membranes during cellular proliferation. The fatty acid component of plasma membranes is comprised of an approximately equal mixture of saturated and unsaturated fatty acids. This ratio of unsaturated to saturated fatty acids is one of the major factors controlling the fluidity and function of plasma membranes, and had been associated with a number of disease states such as diabetes, obesity, cancer, hypertension and atherosclerosis. Since saturated fatty acids are precursors for unsaturated fatty acids, the enzymes that catalyze this conversion, fatty acyl-CoA desaturases, must be tightly regulated to maintain the proper fatty acid ratios in the membrane. The most common unsaturated fatty acid of the plasma membrane is oleate. Oleate is produced from the saturated fatty acid, stearate, by the activity of stearoyl-CoA desaturases (SCD). One potential SCD regulatory mechanism is end-product feedback inhibition by oleate. It has been well demonstrated that in lipogenic cells, oleate is not a regulator of SCD. However, the primary physiological fate of the product of SCD activity in lipogenic cells is not the plasma membrane as it is in nonlipogenic cells, thus the regulation of SCD in lipogenic cells is likely to be different. Our preliminary data shows this to be the case as oleate strongly inhibits SCO activity in CHO fibroblasts, represses the mRNA levels and transcription of the two known SCD genes, SCD1 and SCD2. The main objective of this proposal is to test the hypothesis that in proliferating cells (as opposed to lipogenic cells) the end product of stearoyl-C0A desaturase activity, oleate, represses the level of SCD mRNAs by inhibiting transcription of the SCD genes. SCD1 and SCD2 are known to be regulated transcriptionally by sterols via sterol response element binding proteins (SREBPs) that bind to sterol response elements (SREs) present in the promoters of both genes. Since we have shown that oleate can repress transcription other promoters containing SREs, we speculate that transcriptional regulation of SCD by oleate will involve SREBPs. The specific aims of the proposal are 1) to identify the role of SREs in oleate regulation of SCD transcription. 2) To determine what trans-acting factors are necessary for oleate regulation of SCD transcription. 3) To determine the mechanism by which oleate represses transcription of SRE containing promoters. Transfection of CHO fibroblasts with SCD reporter genes containing various mutations in the promoters will be used to determine the location of the cis-acting elements that regulate SCD expression in response to oleate. Ectotopic expression of SREBPs or other trans-acting factors will be used to determine their contribution to oleate regulation of SCD transcription.

描述：（从申请人的摘要扫描）长期目标是 了解C18脂肪酸调节合成的机制， 胆固醇和脂肪酸的合成所必需的膜， 细胞增殖。质膜的脂肪酸成分是 由饱和脂肪酸和不饱和脂肪酸的大致相等的混合物组成， acids.这种不饱和脂肪酸与饱和脂肪酸的比例是主要的 控制质膜流动性和功能的因素，并已被 与许多疾病状态如糖尿病，肥胖，癌症， 高血压和动脉粥样硬化。由于饱和脂肪酸是 对于不饱和脂肪酸，催化这种转化的酶，脂肪酸 酰基辅酶A去饱和酶，必须严格调节，以维持适当的脂肪 膜中的酸比例。不饱和脂肪酸是最常见的不饱和脂肪酸。 质膜是油酸盐。油酸由饱和脂肪酸产生， 硬脂酸酯，通过硬脂酰辅酶A去饱和酶（SCD）的活性。一个潜在SCD 调节机制是油酸酯的终产物反馈抑制。已经 充分证明了在脂肪生成细胞中，油酸盐不是SCD的调节剂。 然而，SCD活性产物的主要生理命运在 脂肪细胞不是质膜，因为它是在非脂肪细胞，因此， 脂肪生成细胞中SCD的调节可能不同。我们 初步数据表明，油酸盐强烈抑制SCO， 活性，抑制mRNA水平和转录的CHO成纤维细胞， 两个已知的SCD基因，SCD 1和SCD 2。这项建议的主要目的是 测试假设，在增殖细胞（而不是脂肪细胞） 硬脂酰-C 0A去饱和酶活性的终产物油酸酯抑制 通过抑制SCD基因的转录来降低SCD mRNA水平。SCD 1和SCD 2 已知通过固醇应答由固醇进行转录调节 与固醇反应元件（SRE）结合的元件结合蛋白（SREBP） 存在于两个基因的启动子中。因为我们已经证明油酸可以 抑制转录其他启动子含有SRE，我们推测， 油酸盐对SCD的转录调节将涉及SREBP。具体 该提案的目的是：1）确定SRE在油酸调节中的作用 SCD转录。2)确定哪些反式作用因子是必需的 油酸调节SCD转录。3)通过以下方式确定机制： 所述油酸酯抑制含SRE启动子的转录。转染 具有SCD报告基因的CHO成纤维细胞的细胞周期中含有各种突变， 启动子将用于确定顺式作用元件的位置 其响应油酸调节SCD表达。异位表达 SREBP或其他反式作用因子将用于确定其 油酸调节SCD转录的贡献。