Acyl-CoA Synthetase: Structure, Function and Regulation

DK59935。细胞内脂质，特别是三酰基甘油、脂肪酸、芳基辅酶a及其代谢产物，是肥胖、胰岛素抵抗和糖尿病之间的关键联系。从1990年到1998年，人们克隆了5种不同的大鼠酰基辅酶a合成酶（ACS）同工型，结果表明，不同的ACS同工型可能在调节细胞脂肪酸和酰基辅酶a水平中起主要作用。因此，令人惊讶的是，关于单个ACS亚型的信息很少。使用非交叉反应肽抗体，我们已经证明在肝脏和脂肪细胞中表达的三种异构体ACS1、4和5分别位于肝脏不同的亚细胞膜上，它们受到不同的化学抑制剂的抑制，并且它们在肝脏中通过禁食和再喂养独立调节。此外，我们发现噻唑烷二酮特异性抑制AVCS4，这表明这些临床上重要的胰岛素增敏剂可能部分通过抑制ACS4起作用。我们现在建议将重点放在ACS1、4和5的功能、调控和结构上，以了解这些ACS1、4和5如何促进正常的甘油脂代谢，以及每种ACS异构体在促进胰岛素抵抗和糖尿病的脂质相关病理生理中所起的作用。为了确定ACS的功能，我们将过表达三种ACS亚型，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。我们将用共聚焦显微镜比较ACS的细胞位置，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膜的运动。我们将确定膜内酰基辅酶a合成酶同工异构体的形貌及其晶体结构。最后，我们将使用酵母双杂交系统来确定每个ACS是否有一个或多个特定的代谢伙伴。这些研究将使我们了解酰基辅酶a如何作为代谢信号和合成和能量产生途径的底物，它们如何被分配到不同的代谢命运，以及它们的代谢如何促进糖尿病的发病机制。

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.