White A L, Graham D L, LeGros J, Pease R J, Scott J
Division of Molecular Medicine, Medical Research Council, Harrow, Middlesex, United Kingdom.
J Biol Chem. 1992 Aug 5;267(22):15657-64.
Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.
生理浓度的油酸可刺激HepG2细胞分泌含载脂蛋白（apo）B的脂蛋白，而不会增加apoB mRNA水平。本研究的目的是确定油酸是通过增加apoB mRNA的翻译起作用，还是通过对apoB蛋白的翻译后效应起作用。为了探讨油酸刺激apoB分泌的机制，构建了一系列羧基末端截短的apoB构建体。每个构建体都包含SV40早期启动子、apoB 5'-非翻译区和SV40聚腺苷酸化信号。因此，内源性apoB与构建体编码的蛋白质之间或构建体本身之间对油酸反应的任何差异都应取决于蛋白质序列。在大鼠肝癌细胞系McArdle-RH7777中为每个构建体建立了稳定的转化体。通过用[35S]甲硫氨酸标记、免疫沉淀和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳来确定油酸对apoB蛋白产物分泌的影响。apoB41的羧基末端截短导致apoB分泌对油酸反应的能力丧失。在1.0-1.4 g/ml的连续CsCl梯度上对分泌蛋白进行超速离心，结果表明这与apoB作为浮力脂蛋白颗粒回收能力的降低相关。添加油酸降低了作为脂蛋白分泌的短形式apoB回收时的密度。对apoB100和截短蛋白分泌的脉冲追踪分析表明，它们都经历了快速的翻译后细胞内降解。我们得出结论，油酸对apoB mRNA的翻译没有影响，但通过减少那些能够产生浮力脂蛋白的apoB形式的分泌前降解来促进含apoB脂蛋白的分泌。
