Lewin Tal M, Van Horn Cynthia G, Krisans Skaidrite K, Coleman Rosalind A
Department of Nutrition and Pediatrics, University of North Carolina, Chapel Hill, NC 27599, USA.
Arch Biochem Biophys. 2002 Aug 15;404(2):263-70. doi: 10.1016/s0003-9861(02)00247-3.
Obesity and non-insulin-dependent diabetes favor storage of fatty acids in triacylglycerol over oxidation. Recently, individual acyl-CoA synthetase (ACS) isoforms have been implicated in the channeling of fatty acids either toward lipid synthesis or toward oxidation. Although ACS1 had been localized to three different subcellular regions in rat liver, endoplasmic reticulum, mitochondria, and peroxisomes, the study had used an antibody raised against the full-length ACS1 protein which cross-reacts with other isoforms, probably because all ACS family members contain highly conserved amino acid sequences. Therefore, we examined the subcellular location of ACS1, ACS4, and ACS5 in rat liver to determine which isoform was present in peroxisomes, whether the ACSs were intrinsic membrane proteins, and which ACS isoforms were up-regulated by PPAR alpha ligands. Non-cross-reacting ACS1, ACS4, and ACS5 peptide antibodies showed that ACS4 was the only ACS isoform present in peroxisomes isolated from livers of gemfibrozil-treated rats. ACS4 was also present in fractions identified as mitochondria-associated membrane (MAM). ACS1 was present in endoplasmic reticulum fractions and ACS5 was present in mitochondrial fractions. Incubation with troglitazone, a specific inhibitor of ACS4, decreased ACS activity in the MAM fractions 30-45% and in the peroxisomal fractions about 30%. Because the signal for ACS4 protein in peroxisomes was so strong compared to the MAM fraction, we examined ACS4 mRNA abundance in livers of rats treated with the PPAR alpha agonist GW9578. Treatment with GW9578 increased ACS4 mRNA abundance 40% and ACS1 mRNA 25%. Although we had originally proposed that ACS4 is linked to triacylglycerol synthesis, it now appears that ACS4 may also be important in activating fatty acids destined for peroxisomal oxidation. We also determined that, unlike ACS1 and 5, ACS4 is not an intrinsic membrane protein. This suggests that ACS4 is probably targeted and linked to MAM and peroxisomes by interactions with other proteins.
肥胖和非胰岛素依赖型糖尿病有利于脂肪酸以三酰甘油的形式储存而非氧化。最近，个别酰基辅酶A合成酶（ACS）同工型与脂肪酸向脂质合成或氧化的导向有关。尽管ACS1已定位在大鼠肝脏的三个不同亚细胞区域，即内质网、线粒体和过氧化物酶体，但该研究使用的是针对全长ACS1蛋白产生的抗体，它会与其他同工型发生交叉反应，可能是因为所有ACS家族成员都含有高度保守的氨基酸序列。因此，我们研究了大鼠肝脏中ACS1、ACS4和ACS5的亚细胞定位，以确定哪种同工型存在于过氧化物酶体中，ACS是否为内在膜蛋白，以及哪些ACS同工型被过氧化物酶体增殖物激活受体α（PPARα）配体上调。非交叉反应性的ACS1、ACS4和ACS5肽抗体显示，ACS4是从吉非贝齐处理的大鼠肝脏中分离出的过氧化物酶体中唯一存在的ACS同工型。ACS4也存在于被鉴定为线粒体相关膜（MAM）的组分中。ACS1存在于内质网组分中，ACS5存在于线粒体组分中。用曲格列酮（一种ACS4的特异性抑制剂）孵育后，MAM组分中的ACS活性降低了30 - 45%，过氧化物酶体组分中的ACS活性降低了约30%。由于过氧化物酶体中ACS4蛋白的信号与MAM组分相比非常强，我们检测了用PPARα激动剂GW9578处理的大鼠肝脏中ACS4 mRNA的丰度。用GW9578处理使ACS4 mRNA丰度增加了40%，ACS1 mRNA增加了25%。尽管我们最初认为ACS4与三酰甘油合成有关，但现在看来ACS4在激活 destined for过氧化物酶体氧化的脂肪酸方面可能也很重要。我们还确定，与ACS1和5不同，ACS4不是内在膜蛋白。这表明ACS4可能是通过与其他蛋白质的相互作用而靶向并连接到MAM和过氧化物酶体上的。
