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bThe mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.T he mevalonate pathway leading to cholesterol synthesis is controlled transcriptionally and, for more rapid shutdown, posttranslationally (1). The third step in the pathway, catalyzed by 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), is generally regarded as the rate-limiting step in cholesterol synthesis and has been intensively studied (2, 3). However, squalene monooxygenase (SM) is a neglected rate-limiting enzyme in cholesterol synthesis downstream of HMGCR. A flavin monooxygenase located in the endoplasmic reticulum (ER), SM catalyzes the conversion of squalene into monooxidosqualene (MOS), the step in the mevalonate pathway preceding cyclization to form the steroid backbone. It can also act on its product to yield dioxidosqualene (DOS), the precursor for the potent oxysterol regulator 24(S),25-epoxycholesterol, which fine-tunes acute cholesterol synthesis (4). SM resides after the isoprenoid branch of the mevalonate pathway, committing products to sterol synthesis. This may allow differential control of cholesterol synthesis from that of essential nonsterol products (5).We recently reported that SM's activity is controlled at the posttranslational level via accelerated cholesterol-dependent ubiquitination and proteasomal degradation (5). This regulation requires the first 100 amino acids of the protein-a region that is highly conserved in vertebrates but lacking in lower organisms, such as yeast (Saccharomyces cerevisiae). Moreover, this sequence is sufficient to confer cholesterol-dependent turnover when fused to green fluorescent protein (GFP) (5). We have established that the process of degradation is distinct from the sterol-regulated ubiquitination and proteasomal degradation of HMGCR, as it does not require...

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Oxysterols have long been known for their important role in cholesterol homeostasis, where they are involved in both transcriptional and posttranscriptional mechanisms for controlling cholesterol levels. However, they are increasingly associated with a wide variety of other, sometimes surprising cell functions. They are activators of the Hedgehog pathway (important in embryogenesis), and they act as ligands for a growing list of receptors, including some that are of importance to the immune system. Oxysterols have also been implicated in several diseases such as neurodegenerative diseases and atherosclerosis. Here, we explore the latest research into the roles oxy-sterols play in different areas, and we evaluate the current evidence for these roles. In addition, we outline critical concepts to consider when investigating the roles of oxysterols in various situations, which includes ensuring that the concentration and form of the oxysterol are relevant in that context--a caveat with which many studies have struggled.

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Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

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