Lipid sensing tips the balance for a key cholesterol synthesis enzyme

Squalene monooxygenase (SM) is a rate-limiting enzyme of cholesterol synthesis beyond HMGCR, anchored in the membranes of the endoplasmic reticulum (ER). The first 100 amino acids (SM N100) represent a lipid-responsive degron (degradation signal) capable of sensing two lipids of the cholesterol synthesis pathway in the ER, squalene and cholesterol (, ). The role of the SM N100 degron is simple; it enables SM to be degraded when the catalytic activity of SM is no longer required, stopping the synthesis pathway at this step. When does this phenomenon occur? Cholesterol, the end-product of the pathway, feeds back on SM and accelerates its degradation (right). Thus, excess cholesterol shuts down the synthesis pathway, accumulating squalene (right) (). However, degradation is attenuated when cholesterol levels are low and squalene levels increase (left). It was originally observed that SM levels increased in cells treated with the SM inhibitor NB-598. Further investigation revealed this occurred due to squalene accumulation, which stabilized the SM N100 degron (left) (). Hence, squalene, the substrate of SM, allosterically stabilizes SM, thereby providing a feedforward mechanism to drive cholesterol synthesis and meet the cellular demand for cholesterol (left) (). Importantly, the SM N100 degron is absent in lower organisms such as yeast, which produce ergosterol rather than cholesterol (). From an evolutionary perspective, the degron may have evolved to impart cholesterol-sensing in human SM (). Indeed, transferring the human SM N100 degron to green fluorescent protein enabled cholesterol-mediated degradation (right), whereas the less conserved SM N100 degron from chicken, zebrafish, and lamprey could not impart the same cholesterol-responsiveness (, , ). Mechanistically, squalene stabilizes the SM N100 degron by preventing the binding of MARCH6, an E3 ubiquitin ligase that adds ubiquitin onto the degron (). This subsequently reduces ubiquitination of the degron, a modification typically marking proteins for degradation (). On the other hand, cholesterol acts on the degron by inducing structural changes that facilitate degradation, notably on the reentrant loop and the amphipathic helix (62-QFALFSDILSGL-73) (). Excess cholesterol ejects the spring-loaded amphipathic helix from the ER membrane and misfolds the helix (), initiating the proteasomal degradation of SM by ubiquitination of atypical residues (serines rather than lysines) that flank the amphipathic cholesterol sensor (). Thus, the fate of SM hangs in the balance as it senses two lipids of the pathway through its lipid-responsive degron, ultimately determining the rate at which cholesterol synthesis proceeds.

EQUIPMENT: Odyssey CLx (LI-COR Biosciences)

REAGENTS: V5 Tag Monoclonal Antibody (#R960-25, ThermoFisher Scientific), Anti-α-Tubulin Monoclonal Antibody (#T5168, Sigma-Aldrich), IRDye 800CW Donkey anti-Mouse IgG (H + L) (#926-32212, LI-COR Biosciences)