A paREDOX in the control of cholesterol biosynthesis: Does the NADPH sensor and E3 ubiquitin ligase MARCHF6 protect mammalian cells during oxidative stress by controlling sterol biosynthesis?

Sterols and the reductant nicotinamide adenine dinucleotide phosphate (NADPH), essential for eukaryotic life, arose because of, and as an adaptation to, rising levels of molecular oxygen (O2). Hence, the NADPH and O2-intensive process of sterol biosynthesis is inextricably linked to redox status. In mammals, cholesterol biosynthesis is exquisitely regulated post-translationally by multiple E3 ubiquitin ligases, with membrane associated Really Interesting New Gene (RING) C3HC4 finger 6 (MARCHF6) degrading at least six enzymes in the pathway. Intriguingly, all these MARCHF6-dependent enzymes require NADPH. Moreover, MARCHF6 is activated by NADPH, although what this means for control of cholesterol synthesis is unclear. Indeed, this presents a paradox for how NADPH regulates this vital pathway, since NADPH is a cofactor in cholesterol biosynthesis and yet, low levels of NADPH should spare cholesterol biosynthesis enzymes targeted by MARCHF6 by reducing its activity. We speculate MARCHF6 helps mammalian cells adapt to oxidative stress (signified by low NADPH levels) by reducing degradation of cholesterogenic enzymes, thereby maintaining synthesis of protective cholesterol.

Cholesterol synthesis enzyme SC4MOL is fine-tuned by sterols and targeted for degradation by the E3 ligase MARCHF6.

Cholesterol biosynthesis is a highly regulated pathway, with over 20 enzymes controlled at the transcriptional and posttranslational levels. While some enzymes remain stable, increased sterol levels can trigger degradation of several synthesis enzymes via the ubiquitin-proteasome system. Of note, we previously identified four cholesterol synthesis enzymes as substrates for one E3 ubiquitin ligase, membrane-associated RING-CH-type finger 6 (MARCHF6). Whether MARCHF6 targets the cholesterol synthesis pathway at other points is unknown. In addition, the posttranslational regulation of many cholesterol synthesis enzymes, including the C4-demethylation complex (sterol-C4-methyl oxidase-like, SC4MOL; NAD(P)-dependent steroid dehydrogenase-like, NSDHL; hydroxysteroid 17-beta dehydrogenase, HSD17B7), is largely uncharacterized. Using cultured mammalian cell lines (human-derived and Chinese hamster ovary cells), we show SC4MOL, the first acting enzyme of C4-demethylation, is a MARCHF6 substrate and is rapidly turned over and sensitive to sterols. Sterol depletion stabilizes SC4MOL protein levels, while sterol excess downregulates both transcript and protein levels. Furthermore, we found SC4MOL depletion by siRNA results in a significant decrease in total cell cholesterol. Thus, our work indicates SC4MOL is the most regulated enzyme in the C4-demethylation complex. Our results further implicate MARCHF6 as a crucial posttranslational regulator of cholesterol synthesis, with this E3 ubiquitin ligase controlling levels of at least five enzymes of the pathway.

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.

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.

Twin enzymes, divergent control: The cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post-translationally.

Cholesterol synthesis is a tightly regulated process, both transcriptionally and post-translationally. Transcriptional control of cholesterol synthesis is relatively well-understood. However, of the ∼20 enzymes in cholesterol biosynthesis, post-translational regulation has only been examined for a small number. Three of the four sterol reductases in cholesterol production, 7-dehydrocholesterol reductase (DHCR7), 14-dehydrocholesterol reductase (DHCR14), and lamin-B receptor (LBR), share evolutionary ties with a high level of sequence homology and predicted structural homology. DHCR14 and LBR uniquely share the same Δ-14 reductase activity in cholesterol biosynthesis, yet little is known about their post-translational regulation. We have previously identified specific modes of post-translational control of DHCR7, but it is unknown whether these regulatory mechanisms are shared by DHCR14 and LBR. Using CHO-7 cells stably expressing epitope-tagged DHCR14 or LBR, we investigated the post-translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post-translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates, whereas LBR remained stable. DHCR14 is degraded via the ubiquitin-proteasome system, and we identified several DHCR14 and DHCR7 putative interaction partners, including a number of E3 ligases that modulate DHCR14 levels. Interestingly, we found that gene expression across an array of human tissues showed a negative relationship between the C14-sterol reductases; one enzyme or the other tends to be predominantly expressed in each tissue. Overall, our findings indicate that whereas LBR tends to be the constitutively active C14-sterol reductase, DHCR14 levels are tunable, responding to the local cellular demands for cholesterol.

The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6.

Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.

Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation.

The 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.

Oxysterols: Old Tale, New Twists.

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.

The UPS and downs of cholesterol homeostasis.

An emerging theme in the regulation of cholesterol homeostasis is the role of the ubiquitin proteasome system (UPS), through which proteins are ubiquitylated and then degraded in response to specific signals. The UPS controls all aspects of cholesterol metabolism including its synthesis, uptake, and efflux. We review here recent work uncovering the ubiquitylation and degradation of key players in cholesterol homeostasis. This includes the low-density lipoprotein (LDL) receptor, transcription factors (sterol regulatory element binding proteins and liver X receptors), flux-controlling enzymes in cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase and squalene monooxygenase), and cholesterol exporters (ATP-binding cassette transporters ABCA1 and ABCG1). We explore which E3 ligases are involved, and identify areas deserving of further research.

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting ß-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis.

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.

New insights into cellular cholesterol acquisition: promoter analysis of human HMGCR and SQLE, two key control enzymes in cholesterol synthesis.

BACKGROUND: The two control points of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) are known targets of the transcription factor sterol-regulatory element binding protein-2 (SREBP-2). Yet the location of the sterol-regulatory elements (SREs) and cofactor binding sites, nuclear factor-Y (NF-Y) and specificity protein 1 (Sp1), have not been satisfactorily mapped in the human SQLE promoter, or at all in the human HMGCR promoter. METHODS: We used luciferase reporter assays to screen the sterol-responsiveness of a library of predicted SRE, Sp1 and NF-Y site mutants and hence identify bone fide binding sites. We confirmed SREs via an electrophoretic mobility shift assay (EMSA) and ChIP-PCR. RESULTS: We identified two SREs in close proximity in both the human HMGCR and SQLE promoters, as well as one NF-Y site in HMGCR and two in SQLE. In addition, we found that HMGCR expression is highly activated only when SREBP-2 levels are very high, in contrast to the low density lipoprotein receptor (LDLR), a result reflected in mouse models used in other studies. CONCLUSIONS: Both HMGCR and SQLE promoters have two SREs that may act as a homing region to attract a single SREBP-2 homodimer, with HMGCR being activated only when there is absolute need for cholesterol synthesis. This ensures preferential uptake of exogenous cholesterol via LDLR, thereby conserving energy. GENERAL SIGNIFICANCE: We provide the first comprehensive investigation of SREs and NF-Ys in the human HMGCR and SQLE promoters, increasing our fundamental understanding of the transcriptional regulation of cholesterol synthesis.

The E3 ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters.

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.

Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain.

The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.

The terminal enzymes of cholesterol synthesis, DHCR24 and DHCR7, interact physically and functionally.

Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol "metabolon", where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.

The sterol-based transcriptional control of human 7-dehydrocholesterol reductase (DHCR7): Evidence of a cooperative regulatory program in cholesterol synthesis.

The enzyme 7-dehydrocholesterol reductase (DHCR7) catalyzes the final step of cholesterol synthesis via the Kandutsch-Russell pathway, and is crucial in maintaining cellular cholesterol levels. Its absence leads to the devastating fetal developmental disorder Smith-Lemli-Opitz Syndrome (SLOS). How this enzyme is regulated has implications in controlling not only cholesterol synthesis, but also the synthesis of Vitamin D - another product of 7-dehydrocholesterol. In this study, we look specifically at how DHCR7 is regulated by the sterol regulatory element-binding protein-2 (SREBP-2) transcription factor. Sterol regulation has previously been studied in the rat DHCR7 promoter, but we have found that its regulatory elements are not all conserved in humans. Rather, the human promoter contains two binding sites for SREBP-2 (at -155 and -55) and a binding site for the nuclear factor-Y (NF-Y) cofactor (at -136). The -155 site is a particularly responsive sterol regulatory element (SRE) which is well conserved in mammals, and was possibly overlooked in the rat promoter study. The exact location of the weaker -55 site (close to the known rat SRE) may have shifted during evolution. Furthermore, we established that the two SREs that bind SREBP-2 work in cooperation to synergistically activate DHCR7. We have previously characterized the SREs in DHCR24, the final enzyme in the alternate Bloch pathway of cholesterol synthesis. Here, comparison of the sterol regulation of these terminal enzymes demonstrates the unique cooperative system that helps to control cholesterol synthesis.

Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3ß-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

Controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR).

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the target of the statins, important drugs that lower blood cholesterol levels and treat cardiovascular disease. Consequently, the regulation of HMGCR has been investigated in detail. However, this enzyme acts very early in the cholesterol synthesis pathway, with ∼20 subsequent enzymes needed to produce cholesterol. How they are regulated is largely unexplored territory, but there is growing evidence that enzymes beyond HMGCR serve as flux-controlling points. Here, we introduce some of the known regulatory mechanisms affecting enzymes beyond HMGCR and highlight the need to further investigate their control.

Desmosterol in brain is elevated because DHCR24 needs REST for Robust Expression but REST is poorly expressed.

Cholesterol synthesis in the fetal brain is inhibited because activity of DHCR24 (24-dehydrocholesterol reductase) is insufficient, causing concentrations of the precursor desmosterol to increase temporarily to 15-25% of total sterols at birth. We demonstrate that failure of DHCR24 to be adequately upregulated during periods of elevated cholesterol synthesis in the brain results from the presence in its promoter of the repressor element 1 (RE1) nucleotide sequence that binds the RE1-silencing transcription factor (REST) and that REST, generally reduced in neural tissues, uncharacteristically but not without precedent, enhances DHCR24 transcription. DHCR24 and REST mRNA levels are reduced 3- to 4-fold in fetal mouse brain compared to liver (p < 0.001). Chromatin immunoprecipitation assays suggested that REST binds to the human DHCR24 promoter in the vicinity of the predicted human RE1 sequence. Luminescent emission from a human DHCR24 promoter construct with a mutated RE1 sequence was reduced 2-fold compared to output from a reporter with wild-type RE1 (p < 0.005). Silencing REST in HeLa cells resulted in significant reductions of DHCR24 mRNA (2-fold) and DHCR24 protein (4-fold). As expected, relative concentrations of Δ(24)-cholesterol precursor sterols increased 3- to 4-fold, reflecting the inhibition of DHCR24 enzyme activity. In contrast, mRNA levels of DHCR7 (sterol 7-dehydrocholesterol reductase), a gene essential for cholesterol synthesis lacking an RE1 sequence, and concentrations of HMGR (3-hydroxy-3-methyl-glutaryl-CoA reductase) enzyme protein were both unaffected. Surprisingly, a dominant negative fragment of REST consisting of just the DNA binding domain (about 20% of the protein) and full-length REST enhanced DHCR24 expression equally well. Furthermore, RE1 and the sterol response element (SRE), the respective binding sites for REST and the SRE binding protein (SREBP), are contiguous. These observations led us to hypothesize that REST acts because it is bound in close proximity to SREBP, thus amplifying its ability to upregulate DHCR24. It is likely that modulation of DHCR24 expression by REST persisted in the mammalian genome either because it does no harm or because suppressing metabolically active DHCR24 while providing abundant quantities of the multifunctional sterol desmosterol during neural development proved useful.

Akt phosphorylates Sec24: new clues into the regulation of ER-to-Golgi trafficking.

Regulation of protein transport within the early secretory pathway is a relatively unexplored area. Here, we propose a new player in the control of protein transport from the endoplasmic reticulum (ER) to the Golgi. Akt is an important signaling kinase whose functioning is perturbed in diseases such as cancer and diabetes. We discovered that Akt phosphorylates Sec24, an essential coat protein II (COPII) component involved in mediating cargo selection for ER-to-Golgi trafficking. We discuss how this finding may provide new insights into the regulation of protein transport.