It is this K that likely stabilizes the negatively charged oxygen of the first mevaldyl-CoA intermediate. It is then believed that the close proximity of increases the pKA of E, allowing it to be a proton donor for the reduction of mevaldehyde into mevalonate. As stated before, HMGR is among the most highly regulated enzymes in the human body.
First by regulation of transcription of the reductase gene, which is activated by sterol regulatory element binding protein, a protein that binds to the promoter of the HMGR gene when cholesterol levels fall. The third level of HMGR regulation involves the degradation of intact reductase. Helices of the HMGR transmembrane domain, called the sterol-sensing domain, sense the increased levels of sterols. Additionally, as sterol levels increase, the helices can expose Lysine which can be ubiquitinated and subsequently trigger proteolytic degradation.
SwissPalm i. Bgee i. ExpressionAtlas i. Genevisible search portal to normalized and curated expression data from Genevestigator More Genevisible i. Human Protein Atlas More HPA i.
BioGRID i. ELM i. Protein interaction database and analysis system More IntAct i. BindingDB database of measured binding affinities More BindingDB i. RNAct i. SMR i. Database of comparative protein structure models More ModBase i.
PDBe-KB i. Relative evolutionary importance of amino acids within a protein sequence More EvolutionaryTrace i. Ensembl GeneTree More GeneTree i. InParanoid i. OMA i. Database of Orthologous Groups More OrthoDB i. Database for complete collections of gene phylogenies More PhylomeDB i. TreeFam database of animal gene trees More TreeFam i. Conserved Domains Database More CDD i. Gene3D i. Integrated resource of protein families, domains and functional sites More InterPro i. Pfam protein domain database More Pfam i.
Protein Motif fingerprint database; a protein domain database More Superfamily database of structural and functional annotation More Corresponds to variant dbSNP:rs Ensembl.
The changes in the amino acid sequence may be due to alternative splicing, alternative promoter usage, alternative initiation, or ribosomal frameshifting. Missing in isoform 2. CCDS i. Protein sequence database of the Protein Information Resource More PIR i. RefSeq i. Ensembl eukaryotic genome annotation project More Ensembl i.
GeneID i. Statins are the most prescribed drug regimen for treating CVDs since they were first introduced to the market in the late s. Accompanying with the cardiovascular benefits, however, all kinds of statins block the generation of mevalonate derivatives that inhibit HMGCR expression through multiple feedback regulations, causing a remarkable increase of the enzyme Fig.
This, as a result, hampers the effectiveness of the drug, provoking more intensive treatments that elicit unfavorable side effects 25 , 26 , 27 , 28 , Our study provides a potential solution to this problem. In this study, we have successfully identified the cholesterol derivative Cmpd 81 that promotes the degradation of HMGCR without activating the lipogenic activity of LXR. It can function alone as well as potentiate the efficacy of lovastatin in ameliorating cholesterol diet-induced hyperlipidemia and reducing atherosclerotic plaques.
This study proves the concept that inducing HMGCR degradation can effectively reduce serum cholesterol levels and overcome the defect of statins Fig.
A working model of Cmpd 81 in lowering cholesterol synthesis. However, Cmpd 81 remarkably degrades statins-induced HMGCR protein through the ubiquitin-proteasome pathway to lower the cholesterol synthesis, and to improve the efficacy of statins. Recent studies indicate that hepatic accumulated free cholesterol is a critical lipotoxic molecule in the development of nonalcoholic steatohepatitis NASH , as the increased hepatic cholesterol synthesis and expression of HMGCR are associated with the severity of NASH 53 , Moreover, statin usage was reported to reduce hepatic steatosis, inflammation and fibrosis in NASH patients Thus, it will be interesting to evaluate the therapeutic effects of Cmpd 81 and statin, alone or synergistically, on the liver inflammation and fibrosis in NASH models.
In addition, considering the pleiotropic effects of statin therapy related to the cholesterol lowering and mevalonate-derived isoprenoids depletion, it is reasonable to explore the combinatory effects of statin and Cmpd 81 in the conditions where statins alone are beneficial, such as cancer 56 , central nervous system pathology 57 , pulmonary alveolar proteinosis 58 , vaccine adjuvant 59 , and even cancer immunotherapy The design of compounds in our study was fueled by the knowledge of the molecular mechanisms of HMGCR degradation 13 , Cholesterol derivatives, including oxysterols e.
We found that, in consistent with previous findings 12 , di-methylation at C4 is important for sterol-regulated HMGCR degradation. Cmpd 81, one of the most effective chemicals, shows an EC 50 value approximately fold and 4-fold more potent than that of cholesterol and 24,DHL, respectively. Further optimization on the chemical structure of Cmpd 81 may improve its activity and other drug-related properties.
Originated from a natural cellular metabolite with a physiologically validated mechanism, Cmpd 81 or this class of compounds is certainly advantageous at least in two aspects. Secondly, unlike lanosterol and 24,DHL that are sterol intermediates of the cholesterol biosynthetic pathway, Cmpd 81 cannot be converted to cholesterol. The safety profile of Cmpd 81 has also been evaluated. Administration of Cmpd 81 for up to 20 weeks in mice did not affect body weight and food intake.
It appears to be safe and efficacious to target HMGCR for degradation, and feasible for further drug development. Moreover, we evaluated the preclinical cardiac safety of Cmpd The early preclinical cardiac safety assessment is a major concern in the drug discovery and development, given that many non-cardiovascular drugs e.
Human ether-a-go-go- related gene HERG encodes heart highly expressed hERG potassium channels, and hERG current blockages are recognized as the predominant mechanism of drug-induced cardiac arrhythmias These data indicate that Cmpd 81 would be a good drug candidate without hERG channel associated cardiac toxicity.
Collectively, we have synthesized and characterized a series of compounds that effectively induces HMGCR degradation based on structure—activity relationship studies. Using Cmpd 81 as a representative, we prove the concept that this class of chemicals can prevent statins-induced accumulation of HMGCR, reduce serum cholesterol levels and decrease atherosclerosis. Our work suggests that inducing HMGCR degradation by Cmpd 81 or other chemicals can be a promising strategy alone or synergetic with statin therapy for the treatment of hyperlipidemia and atherosclerosis.
Lipoprotein-deficient serum LPDS 63 and delipidated fetal calf serum 64 were prepared from fetal bovine serum FBS S, Biowest by ultracentrifugation in our laboratory. Horseradish peroxidase-conjugated goat anti-mouse , and anti-rabbit , secondary antibodies were from Jackson ImmunoResearch Laboratories. The primers used are listed in Supplementary Table 1. The cell lines have not been authenticated recently. No test for mycoplasma contamination was performed.
Surviving colonies were isolated and confirmed for GFP expression by immunoblotting and immunofluorescence. CHG cells were treated as described in figure legends.
Briefly, cells were seeded at day 0 in well black plate , Corning at a density of 1. Five random selected filed per well were captured, and about cells were used to determine the mean cytoplasmic GFP fluorescent intensity per cell after subtracting background GFP intensity.
The half-maximal effective concentration EC 50 values were determined using nonlinear regression equation: log inhibitor vs response variable slope, four parameters by Prism 6 software GraphPad. The protein concentrations of lysates were determined using BCA method Beyotime. Samples were mixed with the membrane protein solubilization buffer Uncropped blots are shown in Supplementary Figs.
The supernatant was removed to a new tube, and centrifuged at 1. The pellet was suspended in 0. The suspension was further centrifuged at 1. Samples were finally subjected to immunoblotting as described above. To assay for the ubiquitination of overexpressed HMGCR protein, cells were first transfected with indicated plasmids. The primers used were listed in Supplementary Tables 2 — 4. Mice had ad libitum access to water and standard chow diet SLAC unless mentioned otherwise.
Mice were gavaged as described in figure legends with saline containing 0. Mice were anesthetized and blood collection was performed. Livers were harvested and homogenized in a mixture of chloroform and methanol using Precellys 24 Bertin. The organic phase was dried under N 2 and liver lipids were reconstituted in ethanol. Fractions were eluted with PBS at a constant rate of 0.
The cholesterol distribution curves of lipoproteins were done with GraphPad Prism 6 software. Then the tested compound was applied for 2. The last three peak tail current values of each period treated with compounds were quantified with GraphPad Prism 6 software to determinate the IC 50 values. All statistical analyses were performed using the GraphPad Prism 6 software. Statistical tests were justified as appropriate for every figure.
Sample sizes, statistical tests and P values for each experiment are depicted in the relevant figure legends. Experiments on cultured cells were successfully repeated for three times.
Experiments on mice were performed once with indicated n and biological replicates. The synthesis procedure and spectra characterization of compounds used in this study are described in the Supplementary Figs.
Further information on research design is available in the Nature Research Reporting Summary linked to this article. The data that support the findings of this study are available within the paper and its supplementary information files, and from the corresponding author upon reasonable request. Benjamin, E. Heart disease and stroke statistics update: a report from the American Heart Association.
Circulation , e—e Article Google Scholar. Delahoy, P. The relationship between reduction in low-density lipoprotein cholesterol by statins and reduction in risk of cardiovascular outcomes: an updated meta-analysis.
Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from , participants in 26 randomised trials. Lancet , — Baigent, C. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90, participants in 14 randomised trials of statins. Scandinavian Simvastatin Survival Study Group.
Randomised trial of cholesterol lowering in patients with coronary heart disease: the Scandinavian Simvastatin Survival Study 4S. Google Scholar. Long-term effectiveness and safety of pravastatin in patients with coronary heart disease and average cholesterol concentrations: the LIPID trial follow-up.
Heart Protection Study Collaborative Group. Lancet , 7—22 Istvan, E. Science , — Corsini, A. Scap-Insig binding is mediated by a segment of Scap's membrane domain that includes transmembrane helices 25 , A similar stretch of transmembrane helices is found in at least four other polytopic membrane proteins including the Niemann Pick C1 protein, Patched, Dispatched, and reductase , all of which have been postulated to interact with sterols.
Thus, the region has become known as the sterol-sensing domain The importance of the sterol-sensing domain in regulation of Scap is illustrated by findings that point mutations within the region disrupt Insig binding, thereby relieving mutant Scap-SREBP complexes from sterol-mediated ER retention 30 , 31 , 34 , 35 , The recognition of sequence resemblances between the sterol-sensing domains of Scap and reductase stimulated an appraisal of a role for Insigs in degradation of reductase.
This effort led to the following observations, which considered together divulge the action of at least one of the Insig proteins in sterol-accelerated degradation of reductase.
First, when overexpressed by transfection in Chinese hamster ovary CHO cells, reductase cannot be degraded when the cells are treated with sterols Co-expression of Insig-1 restores sterol-accelerated degradation of reductase, suggesting the saturation of endogenous Insigs by the overexpressed reductase. Degradation of reductase coincides with sterol-induced binding of its membrane domain to Insigs 37 , an action that requires a tetrapeptide sequence, YIYF, located in the second transmembrane segment of reductase see Figure 2B A mutant form of reductase in which the YIYF sequence is mutated to alanine residues no longer binds to Insigs and the enzyme is not subject to rapid degradation.
The YIYF sequence is also present in the second transmembrane domain of Scap, where it mediates sterol-dependent formation of Scap-Insig complexes 30 , In fact, overexpressing the sterol-sensing domain of Scap in cells blocks Insig-mediated, sterol-accelerated degradation of reductase.
This indicates that Scap and reductase bind to the same site on Insigs and the two proteins compete for limiting the amount of Insigs when intracellular sterol levels rise. Evidence supporting a major role for the ubiquitin-proteasome pathway in sterol-accelerated degradation of reductase was first provided by the observation that proteasome inhibition blocks the process 19 , leading to the accumulation of ubiquitinated forms of reductase on ER membranes 9.
This ubiquitination is obligatory for degradation of reductase and exhibits an absolute requirement for the presence of Insigs 38 , Reduction of Insig-1 and Insig-2 mRNA by genetic mutation or RNAi-mediated knockdown abrogates sterol-dependent ubiquitination of endogenous reductase, rendering the enzyme refractory to accelerated degradation. Moreover, sterol-induced ubiquitination of reductase exhibits an absolute Insig requirement in transient transfection assays.
Mutation of the YIYF sequence in reductase, which blocks Insig binding, prevents regulated ubiquitination and slows the enzyme's degradation. In contrast, conservative substitutions of arginine for lysines 89 and in the membrane domain of reductase Figure 2B do not block Insig binding, but the substitutions rather abolish ubiquitination and subsequent degradation of reductase. Thus, lysines 89 and in reductase are implicated as sites for Insig-mediated, sterol-induced ubiquitination.
It is important to note that mutation of lysines 89 and blocks ubiquitination and degradation of reductase in the context of the full-length enzyme, suggesting that the catalytic domain does not contribute to ubiquitination.
This is consistent with the observation that the soluble catalytic domain is dispensable for sterol-regulated degradation How might Insig binding impart recognition of reductase by the ubiquitinating machinery?
This question was addressed by examining reductase ubiquitination in a permeabilized cell system Sterol-depleted cells were permeabilized with low concentrations of the mild detergent digitonin such that nearly all of the cytosolic proteins were released into the supernatant upon centrifugation, whereas membrane proteins such as reductase remained associated with the pellet fraction.
The pellet of permeabilized cells supports Insig-dependent ubiquitination of reductase that is stimulated by additions of ATP, sterols, and rat liver cytosol in vitro.
Surprisingly, reductase ubiquitination is potently stimulated by oxygenated derivatives of cholesterol, including , , and hydroxycholesterol, but not by cholesterol itself. The significance of this finding will be discussed in more detail below.
Ubiquitination of proteins is a multistep process, involving the action of at least three types of enzymes In the first step, ubiquitin is activated by the ubiquitin-activating enzyme E1 , which forms a thiol ester between a reactive cysteine residue in E1 and the C-terminus of ubiquitin.
Next, ubiquitin is transferred from E1 to a catalytic cysteine of the ubiquitin-conjugating enzyme E2. The third type of enzyme, ubiquitin ligase E3 , facilitates transfer of activated ubiquitin from E2 to a lysine residue in the substrate or a previously attached ubiquitin. Once a poly-ubiquitin chain of sufficient size is built, the substrate is recognized and subsequently degraded by proteasomes.
Only two E1 enzymes exist, and both are cytosolic proteins. In contrast, a variety of E2s and E3s, both soluble and membrane-bound, have been described The exquisite sensitivity of substrate ubiquitination is ultimately determined by the E3, either alone or in combination with its cognate E2. Fractionated S from Hela cells was utilized to determine which component of the reductase ubiquitinating machinery E1, E2 and E3 is provided by rat liver cytosol in the permeabilized cell system These fractions were first described by Hershko and co-workers 43 , 44 and were generated by separating Hela cell S into fractions that bind Fraction II or do not bind Fraction I an anion exchange resin.
Fraction II effectively replaces rat liver cytosol for regulated ubiquitination of reductase in permeabilized cells, but Fraction I does not. Two observations indicate that Fraction II provides a source of ubiquitin activation in the permeabilized cell system. First, purified E1 replaces rat liver cytosol for sterol-regulated ubiquitination of reductase in permeabilized cells.
Second, immunodepletion of E1 eliminates the reductase ubiquitinating activity of rat liver cytosol. These results demonstrate that E1 is the only cytosolic protein required for reductase ubiquitination, which indicates the reductase E2 and E3 are membrane-associated proteins. This notion is consistent with the localization of apparent sites of reductase ubiquitination, lysines 89 and , which are cytosolically exposed and are predicted to lie immediately adjacent to transmembrane helices three and seven Figure 2B.
Results from the analysis of reductase ubiquitination in permeabilized cells indicated that Insig binding results in recruitment of enzymes that ubiquitinate reductase. Coimmunoprecipitation experiments, coupled with tandem mass spectroscopy, were utilized to identify membrane proteins that associate with the sterol-dependent reductase-Insig complex.
These studies revealed that Insig-1 binds to a known membrane-anchored ubiquitin ligase called gp78 The cDNA for gp78 predicts a amino acid protein that can be divided into four domains. The N-terminal domain of amino acids contains five to seven membrane-spanning helices that anchor the protein to ER membranes and mediate association with Insig The membrane attachment region of gp78 is followed by a amino acid region with a RING finger consensus sequence that confers ubiquitin ligase activity Following the RING domain is a amino acid region homologous to Cue1p, an ER membrane protein in yeast that serves as a membrane anchor for Ubc7p, a cytosolic ubiquitin-conjugating enzyme Recently, this region of gp78 has been shown to directly bind to Ufd1, a cytosolic protein that modulates gp78 ubiquitin ligase activity, thereby enhancing ubiquitination and degradation of the enzyme's substrates At least three lines of evidence indicate that gp78, through its binding to Insig-1, initiates sterol-accelerated degradation of reductase.
Importantly, the effect of gp78 knockdown is specific inasmuch as knockdown of a related membrane-bound ubiquitin ligase, Hrd1, does not affect reductase ubiquitination. Another function of gp78, besides its role as a ubiquitin ligase, is to couple ubiquitination of reductase to degradation through its association with VCP.
Indeed, coimmunoprecipitation experiments show that gp78 is an intermediary in association of VCP and Insig The identification of gp78 as an E3 ubiquitin ligase that mediates reductase ubiquitination has important implications for yet another mode of sterol regulation.
The regulation of Insig-1 contrasts that of reductase in that Insig-1 becomes ubiquitinated and is rapidly degraded by proteasomes in sterol-depleted cells Ubiquitination of Insig-1 is mediated by gp78 When sterols induce reductase to bind Insig-1, ubiquitination is diverted toward reductase and the enzyme becomes rapidly degraded. However, when sterols cause Scap to bind Insig-1, gp78 is displaced and no longer ubiquitinates Insig-1, thereby stabilizing the protein.
This reaction helps to explain why reductase is degraded when it binds to Insig-1, whereas Scap binding to Insig-1 leads to retention in the ER.
Increased transcription of the Insig-1 gene leads to increased synthesis of Insig-1 protein, but the protein is ubiquitinated and degraded until sterols build up to levels sufficient to trigger Scap binding.
Oxysterols are derivatives of cholesterol that contain hydroxyl groups at various positions in the iso-octyl side chain 53 , These compounds are synthesized in many tissues by specific enzymes called hydroxylases; oxysterols play key roles in cholesterol export and they are also intermediates in the synthesis of bile acids Oxysterols are significantly more soluble than cholesterol in aqueous solution, and thus can readily pass across the plasma membrane and enter cells.
This property renders oxysterols such as , , and hydroxycholesterol extremely potent in inhibiting cholesterol synthesis by stimulating binding of both reductase and Scap to Insigs. Oxysterols are present at very low concentrations 10 4 - to 10 6 -fold less than cholesterol in tissues and blood, which raises questions as to whether they act through a similar mechanism as LDL-derived cholesterol to block cholesterol synthesis.
In the case of Scap, the mode of action of these two classes of sterols is becoming clear. Cholesterol directly binds to the membrane domain of Scap in a specific and saturable fashion The interaction causes a conformational change in Scap that promotes Insig binding The addition of cholesterol in vitro to membranes isolated from sterol-depleted cells causes exposure of a cryptic trypsin cleavage site, thereby altering the tryptic digestion pattern of Scap that can be monitored by immunoblot analysis Co-expression of Insigs lowers the amount of cholesterol required to induce the conformational change in Scap.
Oxysterols neither alter Scap's conformation in vitro nor bind to the protein's membrane domain, leading to the postulation of the existence of a membrane-bound oxysterol binding protein.
Remarkably, Insig-2 has been recently defined as a membrane-bound oxysterol binding protein with binding specificity that correlates with the ability of oxysterols to inhibit SREBP processing 32 , Thus, formation of the Scap-Insig complex can be initiated by either binding of cholesterol to the membrane domain of Scap or by binding of oxysterols to Insigs. By analogy, the likely mechanism by which oxysterols stimulate degradation of reductase is through their binding to Insigs.
In striking contrast to results obtained with Scap, the analysis of reductase ubiquitination in permeabilized cells revealed that the reaction was potently stimulated by oxysterols, but not by cholesterol These results led to a search for endogenous sterol regulators of reductase ubiquitination and degradation. Previous indirect studies implicated that lanosterol, the first sterol produced in the cholesterol biosynthetic pathway Figure 1 , or one of its metabolites participates in feedback inhibition of reductase.
These observations led to the evaluation of lanosterol and its metabolite 24,dihydrolanosterol as endogenous regulators of reductase ubiquitination and degradation When added to intact cells, lanosterol and 24,dihydrolanosterol potently stimulate ubiquitination and degradation of reductase through a reaction that requires the presence of Insigs.
This is consistent with the inability of lanosterol to directly bind to Scap and Insig or alter Scap's conformation in vitro The action of lanosterol and 24,dihydrolanosterol is direct and does not require their conversion into an active metabolite as indicated by the reconstitution of reductase ubiquitination by simply incubating isolated membranes with the sterols and purified E1. Insig-mediated regulation of reductase is controlled by three classes of sterols: oxysterols, cholesterol, and methylated sterols such as lanosterol and 24,dihydrolanosterol.
Oxysterols, which are derived from cholesterol, have dual actions in that they accelerate degradation of reductase and block ER to Golgi transport of Scap-SREBP through their direct binding to Insigs. Notably, the demethylation of lanosterol has been implicated as a rate-limiting step in the post-squalene portion of cholesterol synthesis, suggesting the reaction as a potential focal point in sterol regulation 63 , Considering that lanosterol is the first sterol produced in cholesterol synthesis, it seems reasonable that it controls early steps in the pathway i.
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