The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Acta Biochim Biophys Sin (Shanghai). 2010 May 15;42(5):303-10. doi: 10.1093/abbs/gmq022.
Sterol-regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is a rapid feedback regulatory mechanism by which cells employ to control the cholesterol biosynthesis. This process is initiated by the sterol-induced interaction between HMGCR and Insig-1/ gp78, a membrane-bound ubiquitin ligase complex. There are two Lys residues (Lys89 and Lys248) facing cytosol in the membrane domain of HMGCR, and Lys248 is the major ubiquitination site. In this study, we investigated the mechanism of ubiquitination site selection in HMGCR. We find that the distance of Lys248 to membrane is dispensable for its ubiquitination. However, the conserved tetra-glutamic acid residues adjacent to Lys248 in HMGCR are essential. Replacement of these negatively charged residues with tetraarginine causes the resistance of HMGCR to sterol-induced ubiquitination and degradation, albeit this mutant HMGCR can still binds to Insig-1. We further find that the tetra-glutamic acid residues are necessary but not sufficient for the modification on their adjacent Lys, since they are not functional on Lys89 of HMGCR or in SCAP. UBE2G2, a previously known E2 of gp78, is demonstrated to be involved in the sterol-regulated ubiquitination and degradation of HMGCR. In summary, these results identify the tetraglutamic acid residues as a critical motif in HMGCR for the ubiquitination reaction mediated by gp78 and UBE2G2.
固醇调节的 3-羟-3-甲基戊二酰辅酶 A 还原酶（HMGCR）降解是细胞用来控制胆固醇生物合成的快速反馈调节机制。这个过程是由固醇诱导的 HMGCR 与 Insig-1/gp78 之间的相互作用启动的，Insig-1/gp78 是一种膜结合的泛素连接酶复合物。HMGCR 膜域中有两个面向细胞质的赖氨酸残基（Lys89 和 Lys248），Lys248 是主要的泛素化位点。在本研究中，我们研究了 HMGCR 泛素化位点选择的机制。我们发现 Lys248 到膜的距离对于其泛素化是可有可无的。然而，HMGCR 中紧邻 Lys248 的保守四谷氨酸残基是必不可少的。用四精氨酸取代这些带负电荷的残基会导致 HMGCR 对固醇诱导的泛素化和降解产生抗性，尽管这种突变型 HMGCR 仍然可以与 Insig-1 结合。我们进一步发现，四谷氨酸残基对于它们相邻的赖氨酸的修饰是必要的，但不是充分的，因为它们在 HMGCR 的 Lys89 或 SCAP 上没有功能。UBE2G2，一种先前已知的 gp78 的 E2，被证明参与了 HMGCR 的固醇调节的泛素化和降解。总之，这些结果确定了四谷氨酸残基是 HMGCR 中 gp78 和 UBE2G2 介导的泛素化反应的关键模体。
