Meigs T E, Roseman D S, Simoni R D
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
J Biol Chem. 1996 Apr 5;271(14):7916-22. doi: 10.1074/jbc.271.14.7916.
We have previously reported that degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in the isoprenoid pathway leading to cholesterol production, can be accelerated in cultured cells by the addition of farnesyl compounds, which are thought to mimic a natural, nonsterol mevalonate metabolite(s). In this paper we report accelerated reductase degradation by the addition of farnesol, a natural product of mevalonate metabolism, to intact cells. We demonstrate that this regulation is physiologically meaningful, shown by its blockage by several inhibitory conditions that are known to block the degradation induced by mevalonate addition. We further show that intracellular farnesol levels increase significantly after mevalonate addition. Based on these results, we conclude that farnesol is a nonsterol, mevalonate-derived product that plays a role in accelerated reductase degradation. Our conclusion is in agreement with a previous report (Correll, C. C., Ng, L., and Edwards, P. A. (1994) J. Biol. Chem. 269, 17390-17393), in which an in vitro system was used to study the effect of farnesol on reductase degradation. However, the apparent stimulation of degradation in vitro appears to be due to nonphysiological processes. Our findings demonstrate that in vitro, farnesol causes reductase to become detergent insoluble and thus lost from immunoprecipitation experiments, yielding apparent degradation. We further show that another resident endoplasmic reticulum protein, calnexin, similarly gives the appearance of protein degradation after farnesol addition in vitro. However, after the addition of farnesol to cells in vivo, calnexin remains stable, whereas reductase is degraded, providing further evidence that the in vivo effects of farnesol are physiologically meaningful and specific for reductase, whereas the in vitro effects are not.
我们之前报道过，3-羟基-3-甲基戊二酰辅酶A还原酶是类异戊二烯途径中导致胆固醇生成的限速酶，在培养细胞中添加法尼基化合物可加速其降解，法尼基化合物被认为是模拟了一种天然的非甾醇甲羟戊酸代谢物。在本文中，我们报道了向完整细胞中添加甲羟戊酸代谢的天然产物法尼醇可加速还原酶降解。我们证明这种调节在生理上是有意义的，已知几种抑制条件可阻断甲羟戊酸添加诱导的降解，这些抑制条件对其的阻断就证明了这一点。我们进一步表明，添加甲羟戊酸后细胞内法尼醇水平显著升高。基于这些结果，我们得出结论，法尼醇是一种由甲羟戊酸衍生的非甾醇产物，在加速还原酶降解中起作用。我们的结论与之前的一份报告（科雷尔，C.C.，吴，L.，和爱德华兹，P.A.（1994年）《生物化学杂志》269，17390 - 17393）一致，该报告使用体外系统研究了法尼醇对还原酶降解的影响。然而，体外观察到的降解刺激似乎是由于非生理过程。我们的研究结果表明，在体外，法尼醇会使还原酶变得不溶于去污剂，从而在免疫沉淀实验中丢失，导致表观降解。我们还表明，另一种内质网驻留蛋白钙连蛋白在体外添加法尼醇后同样会出现蛋白质降解的表象。然而，在体内向细胞中添加法尼醇后，钙连蛋白保持稳定，而还原酶被降解，这进一步证明了法尼醇的体内作用在生理上是有意义的且对还原酶具有特异性，而体外作用并非如此。
