From the School of Biotechnology and Biomolecular Sciences, UNSW Sydney, Sydney, New South Wales 2052, Australia and.
the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520.
J Biol Chem. 2019 Feb 15;294(7):2436-2448. doi: 10.1074/jbc.RA118.005069. Epub 2018 Dec 13.
The E3 ligase embrane-ssociated ing--type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players.
膜相关 E3 连接酶内环-类型手指 6（MARCH6）是一种多结构域酶，与内质网膜结合。它控制着几种已知蛋白底物的水平，包括胆固醇合成中的关键酶——鲨烯单加氧酶。然而，除了自身的自降解外，人们对 MARCH6 本身是如何被调节的知之甚少。使用 CRISPR/Cas9 基因编辑、MARCH6 过表达和免疫印迹，我们在这里发现胆固醇在体内和稳定表达 MARCH6 的 HEK293 细胞中稳定 MARCH6 蛋白。相反，胆固醇缺乏的 HEK293 和 HeLa 细胞失去了以胆固醇依赖的方式降解鲨烯单加氧酶的能力。胆固醇增强 MARCH6 的能力似乎不涉及这种 E3 连接酶中的假定固醇感应结构域，但当用包含 valosin 的蛋白（VCP/p97）进行膜提取或蛋白酶体降解被抑制时，这种能力就会被废除。此外，在两种不能自身降解的 MARCH6 突变体中，胆固醇介导的稳定作用缺失，表明胆固醇通过防止其自降解来稳定 MARCH6 蛋白。用化学伴侣剂进行的实验表明，这可能是通过胆固醇加入后 MARCH6 的构象变化发生的。此外，胆固醇降低了至少三种已知的 MARCH6 底物的水平，表明胆固醇介导的 MARCH6 稳定增加了其活性。我们的发现强调了胆固醇在控制蛋白质水平方面的一个重要新作用，扩展了胆固醇稳态参与者的已知功能谱。
