Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet potentially toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using model cell systems, 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, considered as rate limiting). Cholesterol accelerated the proteasomal degradation of SM which required the N-terminal domain, partially conserved in vertebrates but not in lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol, or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM's N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and suggesting this as a possible control point in cholesterol synthesis.

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...

Akt is a critical regulator of cell growth, proliferation, and survival that is activated by phosphatidylinositol 3-kinase (PI3K). We investigated the effect of PI3K inhibition on activation of sterol regulatory element binding protein-2 (SREBP-2), a master regulator of cholesterol homeostasis. SREBP-2 processing increased in response to various cholesterol depletion approaches (including statin treatment) and this increase was blunted by treatment with a potent and specific inhibitor of PI3K, LY294002, or when a plasmid encoding a dominant-negative form of Akt (DN-Akt) was expressed. LY294002 also suppressed SREBP-2 processing induced by insulin-like growth factor-1. Furthermore, LY294002 treatment down-regulated SREBP-2 or -1c gene targets and decreased cholesterol and fatty acid synthesis. Fluorescence microscopy studies indicated that LY294002 disrupts transport of the SREBP escort protein, SCAP, from the endoplasmic reticulum to the Golgi. This disruption was also shown by immunofluorescence staining when DN-Akt was expressed. Taken together, our studies indicate that the PI3K/Akt pathway is involved in SREBP-2 transport to the Golgi, contributing to the control of SREBP-2 activation. Our results provide a crucial mechanistic link between the SREBP and PI3K/Akt pathways that may be reconciled teleologically because synthesis of new membrane is an absolute requirement for cell growth and proliferation.

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The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.