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Abstract
Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using a variety of CHO cell-lines, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the first rate-limiting enzyme in cholesterol synthesis. At the post-translational level, cholesterol induced the proteasomal degradation of SM, which was reversed by a proteasome inhibitor, eliminating squalene accumulation. The cholesterol accelerated polyubiquitination and proteasomal degradation mechanism for SM is unique from that of HMGR: it is not mediated by Insig, 24,25-dihydrolanosterol or side-chain oxysterols, but rather by cholesterol itself. Furthermore, this mechanism requires the N-terminal domain of SM, which is partially conserved in vertebrates, but not lower organisms. Importantly, the N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins, highlighting its importance as the regulatory domain of SM. This previously unrecognized mechanism underlies the cholesterol-dependent rate-limiting activity of SM, making it an important second control point in cholesterol synthesis.
Owing to its unique location in the cholesterol synthesis pathway, SM also participates in the synthesis of 24(S),25-epoxycholesterol (24,25EC), the only oxysterol to be produced in parallel with cholesterol. 24,25EC acutely regulates the levels of newly synthesized cholesterol. We generated an SM overexpressing stable cell line, and as part of its characterization, show that these cells synthesize relatively more 24,25EC than cholesterol compared to the wild-type cells. This could be attributed to a compensatory mechanism through which these stable cells control the amount of newly synthesized cholesterol. Like SM, 2,3-oxidosqualene cyclase (OSC) also participates in the synthesis of 24,25EC and cholesterol, and when partially inhibited, OSC produces more 24,25EC. Using this approach, we show that in cultured cells, increased 24,25EC levels helps overcome the Statin Rebound Effect , a phenomenon characterized by a burst in cholesterol synthesis due to the upregulation of the cholesterol synthesis pathway post-statin treatment.