Degron features of the regulatory domain of squalene monooxygenase - a rate limiting enzyme in cholesterol synthesis

Abstract

Cholesterol is an essential lipid associated with many important biological functions. At both the cellular and physiological levels, cholesterol is acquired through two main sources. One source is uptake, while the other source is de novo cholesterol synthesis. Squalene monooxygenase (SM) is a rate-limiting enzyme of cholesterol synthesis. A few studies have suggested SM could be a promising treatment target to lower cholesterol levels in the blood and as another metabolic target in certain cancers. Thus, there is an increasing need to understand the regulation of this enzyme. One critical mode of regulation is the cholesterol-accelerated degradation of SM. This process requires the first 100 amino acids of SM (termed SM N100). The SM N100 regulatory domain represents a degron region (a degradation signal), which allows SM to be regulated by cholesterol. However, insights into cholesterol sensing by SM N100 and the mechanisms by which SM N100 confers instability were unknown. To investigate this degron, we utilised SM N100 fused to green fluorescent protein, a fusion protein which recapitulates the cholesterol-accelerated degradation of SM. Here, we have performed a series of point mutations, truncations and domain swaps based on our understanding of known degron features. We identified that an amphipathic helix (residues Gln62–Leu73) in SM N100 is required for cholesterol-accelerated degradation. We also present evidence that the cholesterol-driven disorder of the amphipathic helix lengthens the disordered region surrounding the helix and exposes a hydrophobic patch which accelerates SM N100 degradation. Attempts to identify ubiquitination sites revealed SM N100 undergoes non-canonical ubiquitination at serine residues to signal SM N100 for degradation. Finally, we identified valosin-containing protein (VCP) as a key protein which mediates the removal of the SM N100 degron from the endoplasmic reticulum into the cytosol for degradation. In summary, we have increased our understanding of the SM N100 degron architecture, furthering insights into how cholesterol sensing in the endoplasmic reticulum is coupled to protein quality control.