Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol
Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol
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DOI：
10.1074/jbc.m310053200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
DeBose-Boyd, RA
中科院分区：
文献类型：
--
作者：
Sever, N;Song, BL;DeBose-Boyd, RA
内质网酶3-羟基-3-甲基戊二酰辅酶A还原酶产生甲羟戊酸，甲羟戊酸转化为甾醇和其他产物，包括连接在蛋白质上的香叶基香叶醇基团。已知当甲羟戊酸代谢的甾醇和非甾醇终产物在细胞中积累时，该酶被泛素化并迅速降解。在这里，我们使用RNA干扰表明，甾醇加速泛素化的还原酶需要Insig-1和Insig-2，膜结合的蛋白质的内质网，以前被证明加速降解的还原酶时，过表达的转染。丙氨酸取代实验表明，还原酶与Insigs的结合和随后的泛素化需要还原酶第二跨膜螺旋中的四肽序列YIYF。YIYF肽也存在于SCAP的甾醇敏感结构域中，SCAP是另一种以甾醇刺激方式与Insigs结合的蛋白质。当还原酶的赖氨酸248被精氨酸取代时，Insig结合持续存在，但还原酶不再被遍在蛋白化，降解显着减慢。赖氨酸248被预测位于紧邻跨膜螺旋，这表明膜结合的泛素转移酶负责。最后，我们表明，Insig依赖，甾醇刺激的还原酶降解进一步加速时，细胞也提供了20-碳类异戊二烯香叶基香叶醇，但不是15-碳法尼醇，提高还原酶调节的非甾醇增效剂是香叶基香叶基化蛋白的可能性。
The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl-CoA reductase produces mevalonate, which is converted to sterols and to other products, including geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and rapidly degraded when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Here, we use RNA interference to show that sterol-accelerated ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the endoplasmic reticulum that were shown previously to accelerate degradation of reductase when overexpressed by transfection. Alanine substitution experiments reveal that binding of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a sterol-stimulated fashion. When lysine 248 of reductase is substituted with arginine, Insig binding persists, but the reductase is no longer ubiquitinated and degradation is markedly slowed. Lysine 248 is predicted to lie immediately adjacent to a membrane-spanning helix, suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show that Insig-dependent, sterol-stimulated degradation of reductase is further accelerated when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-carbon farnesol, raising the possibility that the nonsterol potentiator of reductase regulation is a geranylgeranylated protein.