Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.
Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.
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甾醇加速由组成型表达的 cDNA 编码的仓鼠 3-羟基-3-甲基戊二酰辅酶 A 还原酶的降解。
DOI：
10.1128/mcb.5.4.634-641.1985
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发表时间：
1985
影响因子：
5.3
通讯作者：
Luskey,KL
中科院分区：
文献类型：
--
作者：
Chin,DJ;Gil,G;Faust,JR;Goldstein,JL;Brown,MS;Luskey,KL
通过磷酸钙介导的转染将含有仓鼠3-羟基-3-甲基戊二酰辅酶A还原酶全长cDNA的重组质粒引入UT-2细胞，UT-2细胞是中国仓鼠卵巢细胞的突变系，其缺乏3-羟基-3-甲基戊二酰辅酶A还原酶活性，因此需要低密度脂蛋白-胆固醇和甲羟戊酸进行生长。我们选择了一个永久转染的细胞系，命名为TR-36细胞，表达高水平的3-羟基-3-甲基戊二酰辅酶A还原酶活性，因此在低密度脂蛋白和甲羟戊酸的情况下生长。TR-36细胞中还原酶mRNA的组成型合成由猿猴病毒40早期启动子驱动，因此该mRNA不受甾醇类如25-羟基胆固醇或来源于低密度脂蛋白的胆固醇的抑制，当还原酶基因由其自身的启动子驱动时，甾醇类通常抑制还原酶mRNA的转录。虽然TR-36细胞在甾醇存在下继续合成大量的还原酶mRNA和蛋白质，但还原酶活性下降了50 - 60%。这种下降是由于预形成的酶分子的降解速率增加了一倍。目前的数据表明，甾醇加速还原酶蛋白质的降解，而不依赖于对蛋白质合成的任何抑制作用。
A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.