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J Biol Chem. 2019 Feb 15;294(7):2436-2448. doi: 10.1074/jbc.RA118.005069. Epub 2018 Dec 13.

Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.

Author information

1
From the School of Biotechnology and Biomolecular Sciences, UNSW Sydney, Sydney, New South Wales 2052, Australia and.
2
the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520.
3
From the School of Biotechnology and Biomolecular Sciences, UNSW Sydney, Sydney, New South Wales 2052, Australia and aj.brown@unsw.edu.au.

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.

KEYWORDS:

E3 ubiquitin ligase; INSIG-2; RGS2; TEB4; Type 2 iodothyronine deiodinase; cholesterol; post-transcriptional regulation; proteasome; protein degradation; protein stability; squalene monooxygenase

PMID:
30545937
PMCID:
PMC6378986
[Available on 2020-02-15]
DOI:
10.1074/jbc.RA118.005069
[Indexed for MEDLINE]

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