A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis

Abstract

Edited by Dennis R. VoelkerCholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans.

A key mammalian cholesterol synthesis enzyme, squalene monooxygenase, is allosterically stabilized by its substrate

“…The degradation of SM is accelerated by cholesterol, requiring SM-N100 and MARCH6. Previously, we reported that SM senses membrane cholesterol (39), and that the SM-N100 amphipathic helix and reentrant loop undergo conformational changes with cholesterol excess (7,8). Our current work clearly indicates that SM-N100 is more than just a cholesterol sensor (Fig.…”

“…Recent studies have revealed that SM is subjected to negative feedback regulation via accelerated degradation under cholesterol-rich conditions. SM senses excess cholesterol in the endoplasmic reticulum (ER) membrane through its N-terminal 100-residue regulatory region (SM-N100), and alters its own stability depending on the cholesterol concentration (5,7,8). Membrane-associated ring-CH type finger 6 (MARCH6) is the cognate E3 ligase for SM (9,10), catalyzing its ubiquitination in the N100 region (11).…”

“…Despite its importance in cholesterol-accelerated degradation, the structure and function of the SM-N100 region has not been fully resolved. Thus far, biochemical analyses have revealed the presence of a reentrant loop anchoring SM to the ER membrane and an amphipathic helix required for cholesterol-accelerated degradation (7,8), while X-ray crystallography has revealed the architecture of the catalytic domain of SM (12). However, the highly hydrophobic and disordered nature of the N100 region has hampered purification and structural analysis of this region or full-length SM (12,13).…”

Functional characterization of squalene synthase and squalene epoxidase in Taraxacum koksaghyz

“…Human SQE is regulated by cholesterol‐dependent proteasomal N‐terminal degradation on the ER membrane (Chua, Howe, Jatana, Thukral, & Brown, ; Gill, Stevenson, Kristiana, & Brown, ). In yeast, a similar feedback mechanism is known to be lanosterol‐dependent (Foresti, Ruggiano, Hannibal‐Bach, Ejsing, & Carvalho, ).…”

“…Human SQE is regulated by cholesterol-dependent proteasomal N-terminal degradation on the ER membrane (Chua, Howe, Jatana, Thukral, & Brown, 2017;Gill, Stevenson, Kristiana, & Brown, 2011).…”