The SREBP pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1/2, are membrane-embedded sterol sensors. 25-hydroxycholesterol (25HC)-dependent association of Scap and Insigs acts as the master switch for the SREBP pathway. Here, we present cryo-EM analysis of the human Scap and Insig-2 complex in the presence of 25HC, with the transmembrane (TM) domains determined at an average resolution of 3.7 Å. The sterol sensing domain (SSD) in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4-S6 segments in Scap and TMs 3/4 in Insig-2 in the luminal leaflet of the membrane. Unwinding of the middle of the Scap-S4 segment is crucial for 25HC binding and Insig association.

Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.

Please cite this article as: Coates HW, Capell-Hattam IM, Brown AJ, The mammalian cholesterol synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form,

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The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.