Jia Zhenzhen, Moulson Casey L, Pei Zhengtong, Miner Jeffrey H, Watkins Paul A
Kennedy Krieger Institute, Johns Hopkins University School of Medicine, 707 N. Broadway, Baltimore, MD 21205, USA.
J Biol Chem. 2007 Jul 13;282(28):20573-83. doi: 10.1074/jbc.M700568200. Epub 2007 May 23.
Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54-64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.
脂肪酸转运蛋白4（FATP4）是一种脂肪酰基辅酶A合成酶，它优先将极长链脂肪酸底物（如C24:0）激活为其辅酶A衍生物。为了更深入了解FATP4的生理功能，我们从缺乏FATP4的无皱纹小鼠和野生型（w.t.）小鼠中建立了皮肤成纤维细胞系。通过蛋白质印迹法，FATP4 -/- 成纤维细胞未检测到FATP4蛋白。与w.t. 成纤维细胞相比，缺乏FATP4的细胞中C24:0的激活减少了83%。C24:0的过氧化物酶体降解减少了58%，C24:0掺入主要磷脂种类（减少54 - 64%）、三酰甘油（减少64%）和胆固醇酯（减少58%）的速率显著降低。由于这些脂质代谢过程发生在不同的亚细胞器中，我们使用亚细胞组分的免疫荧光和蛋白质印迹法来研究FATP4蛋白的分布，并测量w.t. 和FATP4 -/- 成纤维细胞组分中的酶活性。FATP4蛋白和酰基辅酶A合成酶活性定位于多个细胞器，包括线粒体、过氧化物酶体、内质网和线粒体相关膜组分。我们得出结论，在小鼠皮肤成纤维细胞中，FATP4是脂质代谢途径中产生极长链脂肪酸辅酶A的主要酶。虽然FATP4缺乏主要影响极长链脂肪酸代谢，但突变的成纤维细胞也表现出荧光长链脂肪酸摄取减少和长链多不饱和脂肪酸水平降低。FATP4缺陷细胞还含有异常的中性脂滴。这些额外的缺陷表明这些细胞中的代谢异常并不局限于极长链脂肪酸。
