Expression and characterization of recombinant rat acyl-CoA synthetases 1, 4, and 5 - Selective inhibition by triacsin C and thiazolidinediones
Expression and characterization of recombinant rat acyl-CoA synthetases 1, 4, and 5 - Selective inhibition by triacsin C and thiazolidinediones
复制标题
DOI：
10.1074/jbc.m010793200
复制
发表时间：
2001-07-06
影响因子：
4.8
通讯作者：
Coleman, RA
中科院分区：
文献类型：
--
作者：
Kim, JH;Lewin, TM;Coleman, RA
三酸甘油酯和曲格列酮对长链脂肪酸掺入细胞脂质的抑制表明存在与特异性代谢途径相关的对底物敏感的和抗性的酰基辅酶A合成酶（ACS，EC 6.2.1.3）。为了验证这一假设，我们克隆并纯化了大鼠ACS 1，ACS 4和ACS 5，存在于肝脏和脂肪细胞中的同种型，在大肠杆菌中表达了ACS-Flag融合蛋白，并通过Flag亲和层析纯化它们。纯化的ACS 1-、4-和8-Flag亚型在ATP的表观K-m值、热不稳定性、最适pH、对Triton X-100的需求以及对N-乙基马来酰亚胺和苯乙二醛的敏感性方面存在差异。ACS抑制剂三胞苷C强烈抑制ACS 1和ACS 4，但不抑制ACS 5。（过氧化物酶体增殖体激活受体γ）配体曲格列酮、罗格列酮和吡格列酮仅强烈且特异性地抑制ACS（4），IC 50小于1.5 μ M。曲格列酮对ACS 4表现出混合型抑制。α-生育酚，其环结构形成曲格列酮的非TZD部分，不抑制ACS 4，表明噻唑烷-2，4-二酮部分是抑制的关键组分。非TZD的PPAR γ配体GW 1929比罗格列酮强7倍，对ACS 1和ACS 4的抑制作用较差，IC 50大于50 μ M，比罗格列酮所需的IC 50高100倍以上，从而证明了TZD抑制的特异性。此外，PPARalpha配体，氯贝特和GW 4647，以及已知掺入复合脂质中的各种异生素羧酸对ACS 1、-4或-5没有影响。这些结果与先前的数据一起表明，与其他代谢途径相比，三酸甘油酯C和曲格列酮强烈抑制三酰甘油合成。表明ACS 1和ACS 4催化用于三酰基甘油合成酰基-Coks的合成，且缺乏三酸甘油酯C对代谢途径的抑制并不能证明缺乏酰基-CoA参与。结果进一步表明，噻唑烷二酮药物的胰岛素增敏作用可能部分通过与ACS 4以不依赖于PPAR γ的方式直接相互作用来实现。
Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC 6.2.1.3) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography, The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1-, 4-, and 8-Flag isoforms differed in their apparent K-m values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal, The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5, The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPAR gamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS(4), with an IC50 of less than 1.5 muM. Troglitazone exhibited a mixed type inhibition of ACS4. alpha -Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPAR gamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC50 of greater than 50 muM, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPAR alpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1, -4, or -5, These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-Coks used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPAR gamma -independent manner.