Abstract
The E3 ligase membrane-associated ring-CH-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.
Highlights
The E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum
Statins target an early step in cholesterol synthesis (catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)3), and their success in treating cardiovascular disease clearly demonstrates the clinical significance of this pathway
We again employed stable overexpression of these mutants, and we found that both were stabilized in the absence of cholesterol, due to the prevention of self-degradation, but neither was further stabilized in the presence of cholesterol (Fig. 7A), indicating that cholesterol likely prevents degradation of MARCH6 mediated by its ubiquitin ligase activity
Summary
We first considered whether MARCH6 gene expression levels are regulated by changing sterol status, as is the case with IDOL [18] and RNF145 [17]. We included the proteasomal inhibitor MG132 for 2 h prior to treatment with cholesterol (in the absence of MG132) This result clearly demonstrated that the MARCH6 protein was increased following cholesterol addition (Fig. 2B), a first with respect to E3 ligases. A different result was found using the general deubiquitinase (DUB) inhibitor PR619; PR619 prevented the increase in MARCH6 protein over its low basal levels in response to cholesterol addition (Fig. 4C). The VCP inhibitor increased MARCH6 protein levels even further than proteasomal inhibition (Fig. 4C) To confirm this result, we employed VCP siRNA and found similar effects (Fig. 4, C and D). We tested stable cell lines expressing MARCH6 with either a Y340A mutation or an L366F mutation, which we predicted would perturb cholesterol regulation by disrupting the putative SSD Both of these mutants were still stabilized by cholesterol (Fig. 5D). Considering that increasing cholesterol status stabilizes MARCH6 protein levels, we
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