Dudek S M, Semenkovich C F
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1995 Dec 8;270(49):29323-9. doi: 10.1074/jbc.270.49.29323.
To better understand the regulation of gene expression by amino acids, we studied the effects of these macronutrients on fatty acid synthase (FAS), an enzyme crucial for energy storage. When HepG2 cells were fed serum-free media selectively deficient in each amino acid, the omission of any single classic essential amino acid as well as Arg or His (essential in some rapidly growing cells) resulted in FAS mRNA levels that were about half of those in complete medium. Control message levels were unaffected and omission of nonessential amino acids did not alter FAS expression. FAS mRNA levels peaked 12-16 h after feeding complete and Ser (nonessential)-deficient media but did not increase in cells fed Lys (essential)-deficient medium. With Lys, FAS mRNA increased over the physiologic concentration range of 15-150 microM, and low concentrations of lysine decreased FAS but not apoB protein mass. Transcription inhibitors mimicked treatment with Lys-deficient media, and nuclear run-off assays showed that Lys-deficient media abolished FAS but not apoB transcription. After treatment with Lys-deficient media, the intracellular Lys pool was rapidly depleted in association with an increase of uncharged (deacylated) tRNA Lys from < 1 to 64% of available tRNA Lys. Even in the presence of the essential amino acid His, increasing the level of uncharged tRNA His with histidinol, a competitive inhibitor of the histidinyl-tRNA synthetase, blocked FAS expression. Tyrosinol treatment did not alter FAS mRNA levels. These results suggest that essential amino acids regulate FAS expression by altering uncharged tRNA levels, a novel mechanism for nutrient control of gene expression in mammalian cells.
为了更好地理解氨基酸对基因表达的调控作用，我们研究了这些常量营养素对脂肪酸合酶（FAS）的影响，FAS是一种对能量储存至关重要的酶。当用选择性缺乏每种氨基酸的无血清培养基培养HepG2细胞时，去除任何一种单一的经典必需氨基酸以及精氨酸或组氨酸（在一些快速生长的细胞中是必需的）都会导致FAS mRNA水平约为完全培养基中的一半。对照信息水平不受影响，去除非必需氨基酸也不会改变FAS的表达。在添加完全培养基和缺乏丝氨酸（非必需氨基酸）的培养基后12 - 16小时，FAS mRNA水平达到峰值，但在添加缺乏赖氨酸（必需氨基酸）的培养基的细胞中并未升高。对于赖氨酸，FAS mRNA在15 - 150微摩尔的生理浓度范围内增加，低浓度的赖氨酸会降低FAS但不会降低载脂蛋白B的蛋白量。转录抑制剂模拟了用缺乏赖氨酸的培养基进行的处理，核转录分析表明，缺乏赖氨酸的培养基消除了FAS的转录，但没有消除载脂蛋白B的转录。在用缺乏赖氨酸的培养基处理后，细胞内赖氨酸池迅速耗尽，同时未负载（去酰化）的赖氨酸tRNA从可用赖氨酸tRNA的<1%增加到64%。即使在存在必需氨基酸组氨酸的情况下，用组氨醇（组氨酰 - tRNA合成酶的竞争性抑制剂）增加未负载的组氨酸tRNA水平也会阻断FAS的表达。酪氨醇处理不会改变FAS mRNA水平。这些结果表明，必需氨基酸通过改变未负载的tRNA水平来调节FAS的表达，这是哺乳动物细胞中营养物质对基因表达控制的一种新机制。
