Acyl-CoA Synthetase: Structure, Function and Regulation

DK59935。细胞内的脂质，特别是三酰甘油、脂肪酸、芳基-COAs及其代谢物，在肥胖、胰岛素抵抗和糖尿病之间起着至关重要的作用。1990-1998年间，我们克隆了5种不同亚型的大鼠酰基辅酶A合成酶(acyl-CoA synthetase，ACS)，很明显，不同的acyl-CoA亚型可能在调节细胞脂肪酸和酰基辅酶A水平方面起主要作用。因此，令人惊讶的是，关于单个ACS亚型的信息很少。利用非交叉反应多肽抗体，我们证明了在肝脏和脂肪细胞中表达的三种异构体ACS1、4和5分别位于肝脏的不同亚细胞膜上，它们被不同的化学抑制剂抑制，并且它们在肝脏中通过禁食和再摄食独立地调节。此外，我们还发现，噻唑烷二酮类化合物可以特异性地抑制AVCS4，这表明这些临床上重要的胰岛素增敏剂可能部分地通过抑制ACS4发挥作用。我们现在建议重点研究ACS1、4和5的功能、调节和结构，以了解每一种ACs如何促进正常的甘油脂代谢，以及每一种ACS亚型在促进胰岛素抵抗和糖尿病的脂质相关病理生理中起什么作用。为了确定ACSS的功能，我们将过度表达三种ACS亚型，并使用选择性的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ACSS的细胞位置，并确定ACS活性的调节是否包括磷酸化/去磷酸化和从胞浆到细胞膜的移动。我们将确定膜内酰辅酶A合成酶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。这些研究将使我们能够理解酰基-COA如何既作为代谢信号又作为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在糖尿病的发病机制中做出贡献。

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.