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...

The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.

There is growing evidence showing that prostate cancer cells have perturbed cholesterol homoeostasis, accumulating cholesterol to promote cell growth. Consequently, cholesterol-lowering drugs such as statins are being evaluated in prostate cancer treatment. Furthermore, natural products such as betulin (from birch tree bark) and tocotrienol (a minor form of vitamin E) have been shown to lower cholesterol levels. Using these drugs and oxysterols, we have determined which aspects of cholesterol homoeostasis should be targeted in prostate cancer, e.g. cellular cholesterol levels are increased by the transcription factor SREBP-2 (sterol-regulatory-element-binding protein isoform 2), whereas LXR (liver X receptor) promotes cholesterol efflux. Whereas betulin exerted non-specific effects on cell viability, tocotrienols produced a strong direct correlation between SREBP-2 activity and cell viability. Mechanistically, tocotrienols lowered SREBP-2 activity by degrading mature SREBP-2 independently of the proteasome. In contrast, no correlation was seen between LXR activity and cell viability, implying that SREBP-2 is a better target than LXR for prostate cancer treatment. Lastly, androgen-dependent and -independent LNCaP cells were both sensitive to tocotrienols. Overall, this suggests that tocotrienols and other drugs targeting the SREBP-2 pathway are a potential therapeutic option for prostate cancer.

Edited by George M. CarmanThe E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players. 3 The abbreviations used are: HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; D2, type 2 iodothyronine deiodinase; MARCH6, membraneassociated RING finger (C3HC4) 6; RGS2, regulator of G protein signaling-2;

Chromatin remodeler complexes exhibit the ability to alter nucleosome composition and positions, with seemingly divergent roles in the regulation of chromatin architecture and gene expression. The outcome is directed by subunit variation and interactions with accessory factors. Recent studies have revealed that subunits of chromatin remodelers display an unexpectedly high mutation rate and/or are inactivated in a number of cancers. Consequently, a repertoire of epigenetic processes are likely to be affected, including interactions with histone modifying factors, as well as the ability to precisely modulate nucleosome positions, DNA methylation patterns and potentially, higher-order genome structure. However, the true significance of chromatin remodeler genetic aberrations in promoting a cascade of epigenetic changes, particularly during initiation and progression of cancer, remains largely unknown.