Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase

• Published in: The Journal of biological chemistry Vol. 294; no. 20; pp. 8134 - 8147
• Main Authors: Chua, Ngee Kiat, Hart-Smith, Gene, Brown, Andrew J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.05.2019; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; CHO Cells; cholesterol; Cholesterol - genetics; Cholesterol - metabolism; cholesterol regulation; Cricetulus; degron; endoplasmic-reticulum-associated protein degradation (ERAD); Enzyme Stability - genetics; Humans; lipid homeostasis; Lipids; Membrane Proteins - genetics; Membrane Proteins - metabolism; membrane-associated ring-CH-type finger 6 (MARCH6); protein degradation; Protein Domains; Protein Structure, Secondary; Proteolysis; SQLE; squalene monooxygenase; Squalene Monooxygenase - genetics; Squalene Monooxygenase - metabolism; ubiquitin; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; membrane-associated ring-CH-type finger 6 (MARCH6); SQLE; cholesterol regulation; lipid homeostasis; cholesterol; squalene monooxygenase; endoplasmic-reticulum-associated protein degradation (ERAD); ubiquitin; protein degradation; degron
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.007798

Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62–Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.