Acyl-CoA Synthetase: Structure, Function and Regulation

酰基辅酶 A 合成酶：结构、功能和调节

• 批准号: 7009913
• 负责人: Rosalind Anne Coleman
• 金额: $ 28.24万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7009913
• 关键词: 3T3 cells; acyl coA; acyltransferase; adipocytes; bioenergetics; carboxylate; cell membrane; cholesterol; confocal scanning microscopy; diabetes mellitus; enzyme activity; enzyme inhibitors; enzyme structure; homeostasis; insulin sensitivity /resistance; isozymes; laboratory rat; lipid metabolism; liver cells; messenger RNA; northern blottings; nucleic acid sequence; obesity; phosphorylation; protein localization; thiazoles; yeast two hybrid system

DK59935. Intracellular lipids, particularly triacylglycerol, fatty acids, aryl-CoAs and their metabolites, provide a critical link between obesity, insulin resistance and diabetes. Between 1990 and 1998 five different rat isoforms of acyl-CoA synthetase (ACS) were cloned and it has become apparent that the different ACS isoforms probably play major roles in regulating cellular fatty acid and acyl-CoA levels. Thus, it is surprising that little information is available about the individual ACS isoforms. Using non-cross-reacting peptide antibodies, we have shown that the three isoforms expressed in liver and adipocytes, ACS1, 4, and 5 are each located in different subcellular membranes in liver, that they are inhibited by different chemical inhibitors, and that they are regulated independently in liver by fasting and refeeding. Further, we discovered that thiazolidinediones specifically inhibit AVCS4, suggesting that these clinically important insulin sensitizers might act, in part, by inhibiting ACS4. We now propose to focus on the function, regulation, and structure of ACS1, 4, and 5 I order to understand how each of these ACSs contributes to normal glycerolipid metabolism an what role each ACS isoform plays in promoting the lipid-related pathophysiology of insulin resistance and diabetes. In order to determine the function of the ACSs , we will over express each of the three ACS isoforms, and use selective chemical and antisense inhibitors to assess effects on synthetic and degradative pathways. We will compare the cellular locations of the ACSs with confocal microscopy and determine whether regulation of ACS activities includes phosphorylation/dephosphorylation and movement from cytosol to intracellular membranes. We will determine the topography of the acyl-CoA synthetase isoforms within membranes and their crystal structures. Finally, we will use the yeast two-hybrid system to determine whether each ACS has one or more specific metabolic partners. These studies will enable us to understand how acyl- CoAs can serve as both metabolic signals and as substrates for synthetic and energy-producing pathways, how they can be partitioned towards different metabolic fates, and how their metabolism contributes to the pathogenesis of diabetes.

DK59935。细胞内脂质，尤其是三酰基甘油，脂肪酸，芳基酸及其代谢物，在肥胖，胰岛素抵抗和糖尿病之间提供了关键的联系。 在1990年至1998年之间，将五种不同的大鼠同工型克隆到克隆中，显然，不同的ACS同工型可能在调节细胞脂肪酸和酰基-COA水平方面起着重要作用。 因此，令人惊讶的是，几乎没有有关单个ACS同工型的信息。 使用非反应肽抗体，我们表明，在肝脏和脂肪细胞中表达的三种同工型ACS1、4和5分别位于肝脏中不同的亚细胞膜中，它们受到不同化学抑制剂的抑制，并且它们受到不同的化学抑制剂的抑制，并且它们在肝脏中被肝脏在肝脏中独立调节。 此外，我们发现噻唑烷二酮特异性抑制了AVCS4，这表明这些临床上重要的胰岛素敏化剂可能部分通过抑制ACS4作用。 现在，我们建议将重点放在ACS1、4和5的功能，调节和结构上，以了解这些ACS中的每一个如何促进正常的甘油代谢，并且每个ACS同工型在促进脂肪相关的胰岛素抵抗和糖尿病的病理生理学中的作用何种作用。 为了确定ACS的功能，我们将超过三种ACS同工型中的每一个，并使用选择性化学和反义抑制剂来评估对合成和降解途径的影响。 我们将将ACS的细胞位置与共聚焦显微镜进行比较，并确定ACS活性的调节是否包括磷酸化/去磷酸化以及从细胞质到细胞内膜的运动。 我们将确定膜及其晶体结构内的酰基-COA合成酶同工型的地形。 最后，我们将使用酵母双杂交系统来确定每个ACS是否具有一个或多个特定的代谢伴侣。 这些研究将使我们能够了解酰基-COA如何用作代谢信号和合成和能源生产途径的底物，如何将它们划分为不同的代谢命运，以及它们的代谢如何对糖尿病的发病机理有效。