The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues.

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examined its lipid droplet-localized pool using complementary immunofluorescence and cell fractionation approaches and found that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of truncated SM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.

Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase.

Cholesterol synthesis is both energy- and oxygen-intensive, yet relatively little is known of the regulatory effects of hypoxia on pathway enzymes. We previously showed that the rate-limiting and first oxygen-dependent enzyme of the committed cholesterol synthesis pathway, squalene monooxygenase (SM), can undergo partial proteasomal degradation that renders it constitutively active. Here, we show hypoxia is a physiological trigger for this truncation, which occurs through a two-part mechanism: (1) increased targeting of SM to the proteasome via stabilization of the E3 ubiquitin ligase MARCHF6 and (2) accumulation of the SM substrate, squalene, which impedes the complete degradation of SM and liberates its truncated form. This preserves SM activity and downstream pathway flux during hypoxia. These results uncover a feedforward mechanism that allows SM to accommodate fluctuating substrate levels and may contribute to its widely reported oncogenic properties.

Cells need cholesterol to work properly but too much cholesterol is harmful and can contribute to atherosclerosis (narrowing of blood vessels), cancer and other diseases. Cells therefore carefully control the activity of the enzymes that are involved in making cholesterol, including an enzyme known as squalene monooxygenase. When the level of cholesterol in a cell rises, a protein called MARCHF6 adds molecules of ubiquitin to squalene monooxygenase. These molecules act as tags that direct the enzyme to be destroyed by a machine inside cells, known as the proteasome, thereby preventing further (unnecessary) production of cholesterol. Previous studies found that squalene monooxygenase is sometimes only partially broken down to make a shorter (truncated) form of the enzyme that is permanently active, even when the level of cholesterol in the cell is high. However, it was unclear what triggers this partial breakdown. The process of making cholesterol uses a lot of oxygen, yet many cancer cells thrive in tumours with low levels of oxygen. Here, Coates et al. used biochemical and cell biology approaches to study the effect of low oxygen levels on the activity of squalene monooxygenase in human cells. The experiments revealed that low oxygen levels trigger squalene monooxygenase to be partially degraded to make the truncated form of the enzyme. Firstly, MARCHF6 accumulates and adds ubiquitin to the enzyme to accelerate its delivery to the proteasome. Secondly, as the proteasome starts to degrade the enzyme, a build-up of squalene molecules impedes further breakdown of the enzyme. This mechanism preserves squalene monooxygenase activity when oxygen levels drop in cells, which may compensate for temporary oxygen shortfalls and allow cells to continue to make cholesterol. Squalene monooxygenase is overactive in individuals with a wide variety of diseases including fatty liver and prostate cancer. Drugs that block squalene monooxygenase activity have been shown to stop cancer cells from growing, but unfortunately these drugs are also toxic to mammals. These findings suggest that reducing the activity of squalene monooxygenase in more subtle ways, such as stopping it from being partially degraded, may be a more viable treatment strategy for cancer and other diseases associated with high levels of cholesterol.

The Non Catalytic Protein ERG28 has a Functional Role in Cholesterol Synthesis and is Coregulated Transcriptionally.

The enzymatic pathway of cholesterol biosynthesis has been well characterized. However, there remain several potential interacting proteins that may play ancillary roles in the regulation of cholesterol production. Here, we identified ERG28 (chromosome 14 open reading frame 1 [C14orf1]), a homologue of the yeast protein Erg28p, as a player in mammalian cholesterol synthesis. ERG28 is conserved from yeast to humans but has been largely overlooked in mammals. Using quantitative RT-PCR, luciferase assays, and publicly available chromatin immunoprecipitation sequencing data, we found that transcription of this gene is driven by the transcription factor SREBP-2, akin to most cholesterol synthesis enzymes, as well as identifying sterol-responsive elements and cofactor binding sites in its proximal promoter. Based on a split luciferase system, ERG28 interacted with itself and two enzymes of cholesterol synthesis (NSDHL and SC4MOL). Huh7 ERG28-KO cell lines were generated, revealing reduced total cholesterol levels in sterol-depleted environments. In addition, radiolabeled metabolic flux assays showed a 60-75% reduction in the rate of cholesterol synthesis in the KO versus wild-type cells, which could be rescued by expression of ectopic ERG28. Unexpectedly, KO of ERG28 also impaired the activation of SREBP-2 under sterol-replete conditions, by a yet-to-be defined mechanism. These results indicate that ERG28 is clearly involved in cholesterol synthesis, although the precise role this noncatalytic protein plays in this complex metabolic pathway remains to be fully elucidated. A deeper understanding of ERG28, and other ancillary proteins of cholesterol synthesis, may help inform therapeutic strategies for diseases associated with aberrant cholesterol metabolism.

The mammalian cholesterol synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form.

Squalene monooxygenase (SM, also known as squalene epoxidase) is a rate-limiting enzyme of cholesterol synthesis that converts squalene to monooxidosqualene and is oncogenic in numerous cancer types. SM is subject to feedback regulation via cholesterol-induced proteasomal degradation, which depends on its lipid-sensing N-terminal regulatory domain. We previously identified an endogenous truncated form of SM with a similar abundance to full-length SM, but whether this truncated form is functional or subject to the same regulatory mechanisms as full-length SM is not known. Here, we show that truncated SM differs from full-length SM in two major ways: it is cholesterol resistant and adopts a peripheral rather than integral association with the endoplasmic reticulum membrane. However, truncated SM retains full SM activity and is therefore constitutively active. Truncation of SM occurs during its endoplasmic reticulum-associated degradation and requires the proteasome, which partially degrades the SM N-terminus and disrupts cholesterol-sensing elements within the regulatory domain. Furthermore, truncation relies on a ubiquitin signal that is distinct from that required for cholesterol-induced degradation. Using mutagenesis, we demonstrate that partial proteasomal degradation of SM depends on both an intrinsically disordered region near the truncation site and the stability of the adjacent catalytic domain, which escapes degradation. These findings uncover an additional layer of complexity in the post-translational regulation of cholesterol synthesis and establish SM as the first eukaryotic enzyme found to undergo proteasomal truncation.

A structure of human Scap bound to Insig-2 suggests how their interaction is regulated by sterols.

The sterol regulatory element-binding protein (SREBP) pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1 and -2, are membrane-embedded sterol sensors. The 25-hydroxycholesterol (25HC)-dependent association of Scap and Insig acts as the master switch for the SREBP pathway. Here, we present cryo-electron microscopy 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 angstrom. The sterol-sensing domain in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4 to S6 segments in Scap and TMs 3 and 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.

Squalene monooxygenase: a journey to the heart of cholesterol synthesis.

Squalene monooxygenase (SM) is a vital sterol synthesis enzyme across eukaryotic life. In yeast, it is a therapeutic target for treating certain fungal infections, and in mammals it is a rate-limiting enzyme that represents a key control point in the cholesterol synthesis pathway. SM introduces an oxygen atom to squalene, which becomes the signature oxygen of the hydroxyl group in cholesterol. Our knowledge of SM has advanced tremendously since its initial cloning and characterization. Early research developed mammalian SM inhibitors to target SM for cholesterol-lowering purposes. The substrate squalene has gained considerable interest for its health benefits and in nanomedicine for delivery of drugs. More recently, SM has been implicated as a key dysregulated component in certain cancers. In this review, we summarize our present knowledge of SM, focusing on the regulation of SM and the gene encoding it, SQLE. Furthermore, we offer insights into the role of SM across different organisms and its significance in human health and disease.

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

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.

Post-translational control of the long and winding road to cholesterol.

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-methylglutaryl CoA 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.

Consulting prostate cancer cohort data uncovers transcriptional control: Regulation of the MARCH6 gene.

Cholesterol accumulation is a hallmark of prostate cancer (PCa) enabled by the upregulation of its synthesis, which presents a potential therapeutic target. This pathway is suppressed by the E3 ubiquitin ligase membrane-associated RING-CH-type finger 6 (MARCH6); however, little is known of MARCH6 regulation, particularly at the transcriptional level. Here, we consulted large transcriptomic PCa datasets to investigate transcription factors and DNA sequence elements that regulate the MARCH6 gene. Amongst 498 primary PCa tissues of The Cancer Genome Atlas, we identified a striking positive correlation between MARCH6 and androgen receptor (AR) gene expression (r = 0.81, p < 1 × 10-117) that held in other primary tumour datasets. Two putative androgen response elements were identified in the MARCH6 gene using motif prediction and mining of publicly accessible chromatin immunoprecipitation-sequencing data. However, MARCH6 expression was not androgen-responsive in luciferase reporter and qRT-PCR assays. Instead, we established that the MARCH6-AR correlation in primary PCa is due to common regulation by the transcription factor Sp1. We located a region 100 bp downstream of the MARCH6 transcriptional start site that contains three Sp1 binding sites and strongly upregulates promoter activity. The functionality of this region, and Sp1-mediated upregulation of MARCH6, was confirmed using pharmacological and genetic inhibition of Sp1. Moreover, modulation of Sp1 activity affected the stability of squalene monooxygenase, a cholesterol biosynthesis enzyme and MARCH6 substrate. We thus establish Sp1 as the first known regulator of the MARCH6 gene and demonstrate that interrogation of transcriptomic datasets can assist in the de novo inference of transcriptional regulation.

Cholesterol, cancer, and rebooting a treatment for athlete's foot.

A key enzyme in cholesterol synthesis is placed firmly on the oncogenic map and demonstrated to be a potential therapeutic target in liver cancer by repurposing a common antifungal agent (Liu et al, this issue).