Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase.

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4-2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to - 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4-2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4-2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4-2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

Role of hydroxymethylglutharyl-coenzyme A reductase in the induction of stem-like states in breast cancer.

PURPOSE: De novo synthesis of cholesterol and its rate-limiting enzyme, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stem-like states in tumors remains unclear. METHODS: A compiled public database from breast cancer (BC) patients was analyzed with the web application SurvExpress. Cell Miner was used for the analysis of HMGCR expression and statin sensitivity of the NCI-60 cell lines panel. A CRISPRon system was used to induce HMGCR overexpression in the luminal BC cell line MCF-7 and a lentiviral pLM-OSKM system for the reprogramming of MCF-7 cells. Comparisons were performed by two-tailed unpaired t-test for two groups and one- or two-way ANOVA. RESULTS: Data from BC patients showed that high expression of several members of the cholesterol synthesis pathway were associated with lower recurrence-free survival, particularly in hormone-receptor-positive BC. In silico and in vitro analysis showed that HMGCR is expressed in several BC cancer cell lines, which exhibit a subtype-dependent response to statins in silico and in vitro. A stem-like phenotype was demonstrated upon HMGCR expression in MCF-7 cells, characterized by expression of the pluripotency markers NANOG, SOX2, increased CD44 +/CD24low/ -, CD133 + populations, and increased mammosphere formation ability. Pluripotent and cancer stem cell lines showed high expression of HMGCR, whereas cell reprogramming of MCF-7 cells did not increase HMGCR expression. CONCLUSION: HMGCR induces a stem-like phenotype in BC cells of epithelial nature, thus affecting tumor initiation, progression and statin sensitivity.

Direct binding to sterols accelerates endoplasmic reticulum-associated degradation of HMG CoA reductase.

The maintenance of cholesterol homeostasis is crucial for normal function at both the cellular and organismal levels. Two integral membrane proteins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and Scap, are key targets of a complex feedback regulatory system that operates to ensure cholesterol homeostasis. HMGCR catalyzes the rate-limiting step in the transformation of the 2-carbon precursor acetate to 27-carbon cholesterol. Scap mediates proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a membrane-bound transcription factor that controls expression of genes involved in the synthesis and uptake of cholesterol. Sterol accumulation triggers binding of HMGCR to endoplasmic reticulum (ER)-localized Insig proteins, leading to the enzyme's ubiquitination and proteasome-mediated ER-associated degradation (ERAD). Sterols also induce binding of Insigs to Scap, which leads to sequestration of Scap and its bound SREBP-2 in the ER, thereby preventing proteolytic activation of SREBP-2 in the Golgi. The oxygenated cholesterol derivative 25-hydroxycholesterol (25HC) and the methylated cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) differentially modulate HMGCR and Scap. While both sterols promote binding of HMGCR to Insigs for ubiquitination and subsequent ERAD, only 25HC inhibits the Scap-mediated proteolytic activation of SREBP-2. We showed previously that 1,1-bisphosphonate esters mimic DHL, accelerating ERAD of HMGCR while sparing SREBP-2 activation. Building on these results, our current studies reveal specific, Insig-independent photoaffinity labeling of HMGCR by photoactivatable derivatives of the 1,1-bisphosphonate ester SRP-3042 and 25HC. These findings disclose a direct sterol binding mechanism as the trigger that initiates the HMGCR ERAD pathway, providing valuable insights into the intricate mechanisms that govern cholesterol homeostasis.

pH-dependent reaction triggering in PmHMGR crystals for time-resolved crystallography.

Serial crystallography and time-resolved data collection can readily be employed to investigate the catalytic mechanism of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl (HMG)-coenzyme-A (CoA) reductase (PmHMGR) by changing the environmental conditions in the crystal and so manipulating the reaction rate. This enzyme uses a complex mechanism to convert mevalonate to HMG-CoA using the co-substrate CoA and cofactor NAD+. The multi-step reaction mechanism involves an exchange of bound NAD+ and large conformational changes by a 50-residue subdomain. The enzymatic reaction can be run in both forward and reverse directions in solution and is catalytically active in the crystal for multiple reaction steps. Initially, the enzyme was found to be inactive in the crystal starting with bound mevalonate, CoA, and NAD+. To observe the reaction from this direction, we examined the effects of crystallization buffer constituents and pH on enzyme turnover, discovering a strong inhibition in the crystallization buffer and a controllable increase in enzyme turnover as a function of pH. The inhibition is dependent on ionic concentration of the crystallization precipitant ammonium sulfate but independent of its ionic composition. Crystallographic studies show that the observed inhibition only affects the oxidation of mevalonate but not the subsequent reactions of the intermediate mevaldehyde. Calculations of the pKa values for the enzyme active site residues suggest that the effect of pH on turnover is due to the changing protonation state of His381. We have now exploited the changes in ionic inhibition in combination with the pH-dependent increase in turnover as a novel approach for triggering the PmHMGR reaction in crystals and capturing information about its intermediate states along the reaction pathway.

Chlorogenic acid regulates the expression of NPC1L1 and HMGCR through PXR and SREBP2 signaling pathways and their interactions with HSP90 to maintain cholesterol homeostasis.

BACKGROUND: Hypercholesterolemia is widely implicated in the etiology of coronary heart disease, stroke, and dementia. Evidence suggests that chlorogenic acid (CA) reduces the risk of cardiovascular disease. PURPOSE: The current study aims to explore the underlying molecular mechanism of CA in lowering cholesterol based on pregnane X receptor (PXR) and sterol regulatory element-binding protein 2 (SREBP2) regulatory pathways and their interactions with heat shock protein 90 (HSP90). METHODS: A hypercholesterolemic mouse model, HepG2 and Caco2 cell models, metabolomics analysis, and co-immunoprecipitation (COIP) were used to study the mechanism of CA lowering cholesterol. RESULTS: Treatment of the hypercholesterolemic mice with CA for 12 weeks significantly reduced body weight, blood lipid, hepatic lipid accumulation, and increased lipid excretion. The nuclear aggregation of PXR and SREBP2 was inhibited simultaneously. In addition, the expression of downstream target genes, including Niemann-pick C1-like 1 (NPC1L1) and 3­hydroxy-3-methylglutaryl-CoA reductase (HMGCR), was downregulated after CA administration. Furthermore, in HepG2 and Caco2 cell models, CA reduced intracellular cholesterol levels by inhibiting the nuclear translocation of PXR and SREBP2 and the expression of NPC1L1 and HMGCR. SREBP2 interacts with PXR through HSP90, and CA reduces the binding stability of SREBP2 and HSP90 and enhances the binding of PXR and HSP90, thus reducing the nuclear accumulation of SREBP2 and PXR simultaneously. Moreover, CA promoted the phosphorylation of AMP-activated protein kinase (AMPK) and its binding to SREBP2. This was not conducive to the binding of HSP90 and SREBP2 but enhanced the binding of HSP90 and PXR, thereby inhibiting the nuclear translocation of SREBP2 and PXR and reducing intracellular cholesterol levels. However, no noticeable direct binding between AMPK and PXR was observed. CONCLUSION: CA downregulates NPC1L1 and HMGCR expression by acting on the AMPK/SREBP2 direct pathway and the AMPK/SREBP2/HSP90/PXR indirect pathway, thus retaining cholesterol homeostasis.

Eugeniin improves cholesterol metabolism in HepG2 cells and Caco-2 cells.

Considering the absence of prior studies on the cholesterol metabolism-improving effects of eugeniin, the present investigation aimed to explore the potential impact of eugeniin on cholesterol metabolism. This study sought to elucidate the molecular mechanisms involved in this process using HepG2 and Caco-2 cells treated with 5 µm eugeniin. The intracellular cholesterol levels in HepG2 and Caco-2 cells were significantly decreased in the 24-h eugeniin-treated group. The protein and messenger ribonucleic acid (mRNA) levels of the low-density lipoprotein receptor (LDLR) were increased, while 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase protein and mRNA levels were decreased in HepG2 cells 6 h of the eugeniin-treated group. Additionally, LDLR protein and mRNA levels were increased in HepG2 cells after 24 h of eugeniin treatment. In Caco-2, the protein and mRNA levels of ATP-binding cassette transporter 1 were increased after 24 h eugeniin treatment. This novel finding indicates that eugeniin improves cholesterol metabolism in human cell cultures.

HMG-CoA reductase degrader, SR-12813, counteracts statin-induced upregulation of HMG-CoA reductase and augments the anticancer effect of atorvastatin.

Statins are cholesterol-lowering drugs that have exhibited potential as cancer therapeutic agents. However, as some cancer cells are resistant to statins, broadening an anticancer spectrum of statins is desirable. The upregulated expression of the statin target enzyme, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase (HMGCR), in statin-treated cancer cells is a well-known mechanism of statin resistance, which can be counteracted by the downregulation of HMGCR gene expression, or degradation of the HMGCR protein. However, the mechanism by which HMGCR degradation influences the anticancer effects of statins remain unreported. We tested the effect of the HMGCR degrader compound SR-12813 at a concentration that did not affect the growth of eight diverse tumor cell lines. Combined treatment with atorvastatin and a low concentration of SR-12813 led to lowering of increased HMGCR expression, and augmented the cytostatic effect of atorvastatin in both statin-resistant and -sensitive cancer cells compared with that of atorvastatin treatment alone. Dual-targeting of HMGCR using statins and SR-12813 (or similar compounds) could provide an improved anticancer therapeutic approach.

Epigenetic suppression of PGC1α (PPARGC1A) causes collateral sensitivity to HMGCR-inhibitors within BRAF-treatment resistant melanomas.

While targeted treatment against BRAF(V600E) improve survival for melanoma patients, many will see their cancer recur. Here we provide data indicating that epigenetic suppression of PGC1α defines an aggressive subset of chronic BRAF-inhibitor treated melanomas. A metabolism-centered pharmacological screen further identifies statins (HMGCR inhibitors) as a collateral vulnerability within PGC1α-suppressed BRAF-inhibitor resistant melanomas. Lower PGC1α levels mechanistically causes reduced RAB6B and RAB27A expression, whereby their combined re-expression reverses statin vulnerability. BRAF-inhibitor resistant cells with reduced PGC1α have increased integrin-FAK signaling and improved extracellular matrix detached survival cues that helps explain their increased metastatic ability. Statin treatment blocks cell growth by lowering RAB6B and RAB27A prenylation that reduces their membrane association and affects integrin localization and downstream signaling required for growth. These results suggest that chronic adaptation to BRAF-targeted treatments drive novel collateral metabolic vulnerabilities, and that HMGCR inhibitors may offer a strategy to treat melanomas recurring with suppressed PGC1α expression.

The role of HMGCR expression in combination therapy of simvastatin and FAC treated locally advanced breast cancer patients.

OBJECTIVE: Several studies have shown the role of statin added to the patient's chemotherapy regimen and the role of Hydroxymethylglutaryl-CoA Reductase (HMGCR) expression in predicting breast cancer patient outcomes. In our previous study, adding statins improved clinical and pathological responses in LABC patients. Furthermore, we planned to study statin's role as a combination to neoadjuvant chemotherapy (NAC) in treating locally advanced breast cancers on the basis of HMGCR expression. Moreover, we aimed to study the association between the patients' clinicopathological characteristics and HMGCR expression. METHODS: This study is a randomized, double-blinded, placebo-controlled trial in two health centers in Indonesia. Each patient enrolled with written informed consent and then randomized to receive either simvastatin 40 mg/day or a placebo, combined with the fluorouracil, adriamycin, and cyclophosphamide (FAC) NAC. RESULTS: HMGCR was associated with low staging and normal serum cholesterol in the high Ki67 level group (p = 0.042 and p = 0.021, respectively). The pre-and post-chemotherapy tumor sizes are significantly correlated in two groups (HMGCR negative expression, p = 0.000 and HMGCR moderate expression, p = 0.001) with a more considerable average decrease in tumor size compared to HMGCR strong expression group. CONCLUSION: Statin therapy might work better in HMGCR-negative or low-expression tumors, although HGMCR expression is associated with better clinical parameters in our study.

3-Hydroxy-3-methylglutaryl coenzyme A reductase genes from Glycine max regulate plant growth and isoprenoid biosynthesis.

Isoprenoids, a large kind of plant natural products, are synthesized by the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. As one of the rate-limiting enzymes in the MVA pathway of soybean (Glycine max), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is encoded by eight isogenes (GmHMGR1-GmHMGR8). To begin, we used lovastatin (LOV), a specific inhibitor of GmHMGR, to investigate their role in soybean development. To further investigate, we overexpressed the GmHMGR4 and GmHMGR6 genes in Arabidopsis thaliana. The growth of soybean seedlings, especially the development of lateral roots, was inhibited after LOV treatment, accompanied by a decrease in sterols content and GmHMGR gene expression. After the overexpression of GmHMGR4 and GmHMGR6 in A. thaliana, the primary root length was higher than the wild type, and total sterol and squalene contents were significantly increased. In addition, we detected a significant increase in the product tocopherol from the MEP pathway. These results further support the fact that GmHMGR1-GmHMGR8 play a key role in soybean development and isoprenoid biosynthesis.

Limb girdle muscular disease caused by HMGCR mutation and statin myopathy treatable with mevalonolactone.

Myopathy is the main adverse effect of the widely prescribed statin drug class. Statins exert their beneficial effect by inhibiting HMG CoA-reductase, the rate-controlling enzyme of the mevalonate pathway. The mechanism of statin myopathy is yet to be resolved, and its treatment is insufficient. Through homozygosity mapping and whole exome sequencing, followed by functional analysis using confocal microscopy and biochemical and biophysical methods, we demonstrate that a distinct form of human limb girdle muscular disease is caused by a pathogenic homozygous loss-of-function missense mutation in HMG CoA reductase (HMGCR), encoding HMG CoA-reductase. We biochemically synthesized and purified mevalonolactone, never administered to human patients before, and establish the safety of its oral administration in mice. We then show that its oral administration is effective in treating a human patient with no significant adverse effects. Furthermore, we demonstrate that oral mevalonolactone resolved statin-induced myopathy in mice. We conclude that HMGCR mutation causes a late-onset severe progressive muscular disease, which shows similar features to statin-induced myopathy. Our findings indicate that mevalonolactone is effective both in the treatment of hereditary HMGCR myopathy and in a murine model of statin myopathy. Further large clinical trials are in place to enable the clinical use of mevalonolactone both in the rare orphan disease and in the more common statin myopathy.

Protective Impact of Vanillic Acid on Lipid Profile and Lipid Metabolic Enzymes in Diabetic Hypertensive Rat Model Generated by a High-Fat Diet.

INTRODUCTION: Diabetes is the most common component of metabolic syndrome, including abdominal obesity, insulin resistance, hypertension, and dyslipoproteinemia. OBJECTIVE: This study aims to determine whether vanillic acid has antihyperlipidemic properties in diabetic hypertensive rats. METHODS: For this study healthy male albino Wister rats (180-220 gm) were selected. A 20-week highfat diet (HFD) was given to produce diabetic hypertension in Wister rats. Control and diabetic hypertensive rats were treated with vanillic acid. Vanillic acid effects on lipid profiles (cholesterol, triglycerides, phospholipids, free fatty acids, high-density lipoproteins (HDL)) and lipid metabolizing enzymes LPL, LCAT, and HMG CoA reductase studied by a conventional method. To understand the effect of vanillic acid control, experimental rat lipid and metabolic enzymes were studied and treated and controlled animal liver tissues were observed using the different histology staining agents. RESULTS: Vanillic acid caused considerable lipid profile reductions except for HDL and increased plasma HDL levels. After eight weeks of vanillic acid administration also boosts lipid marker enzyme activity (HMG CoA reductase, LPL, and LCAT). In addition, vanillic acid reduces the accumulation of collagen in liver tissues. CONCLUSION: These research studies suggest that vanillic acid has antihyperlipidemic effects in diabetic hypertensive rats fed an HFD.

Therapeutic properties and molecular docking study of some phenolic compounds as anti-human lung cancer potential: A biochemical approach.

Chloroxine (5,7-dichloro-8-hydroxyquinoline) is a molecule utilized in some shampoos for the therapy of seborrheic dermatitis of the scalp and dandruff. In this study, we investigated the inhibition effects of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate compounds on the 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA Reductase) and urease enzymes. We have obtained results for the HMG-CoA Reductase and urease enzymes at the micromolar level. In our study, inhibition result of 5,7-dichloro-8-hydroxyquinoline and Methyl 3,4,5-trihydroxybenzoate on HMG-CoA reductase showed lower values 2.28 ± 0.78 and 33.25 ± 5.04 µg/ml, respectively. Additionally, inhibition result of 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate on urease showed lower values 6.18 ± 1.38 and 8.51 ± 1.35 µg/ml, respectively. Molecular docking calculations were made for their biological activities were compared. In the present work, the structures of the related compounds (1 and 2) were drawn using Gaussian 09 software and done geometry optimization at DFT/B3LYP/6-31G* basis set with aforementioned program. Cytotoxicity potential of these compounds against human lung cancer demonstrated that these compounds had good cytotoxic effects. Both compounds significantly decreased lung cell viability from low doses. In addition, 100 µM dose of all compounds caused significant reductions in lung cell viability. In general, we can say that of the two tested compounds, 5,7-dichloro-8-hydroxyquinoline and methyl 3,4,5-trihydroxybenzoate have cytotoxic effects in all cell types, and this effect is particularly strong in lung cells. Activities were performed at concentrations of 10, 20, 50, 70, and 100 µl and we achieved good results. Lung cell viability (%) value was better at 100 µl concentration and IC50 of them were 54.28 and 48.05 µM.

Fine-tuning of nitrogen-containing bisphosphonate esters that potently induce degradation of HMG-CoA reductase.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.

A fungal tolerance trait and selective inhibitors proffer HMG-CoA reductase as a herbicide mode-of-action.

Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.

Post-Translational Regulation of HMG CoA Reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an endoplasmic reticulum (ER)-localized integral membrane protein that catalyzes the rate-limiting step in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). HMGCR is subjected to strict feedback control through multiple mechanisms to ensure cells constantly produce essential nonsterol isoprenoids, but do not overaccumulate cholesterol. Here, we focus on the mechanism of feedback control of HMGCR that involves its sterol-induced ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We will also discuss the how GGpp-regulated intracellular trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) inhibits HMGCR ERAD to balance the synthesis of sterol and nonsterol isoprenoids. Finally, we will summarize various mouse models, the characterization of which establish that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMGCR and cholesterol metabolism in vivo.

Regulated degradation of HMG CoA reductase requires conformational changes in sterol-sensing domain.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2-4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2-6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations-one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.

Role of BLACAT1 in IL-1ß-Induced Human Articular Chondrocyte Apoptosis and Extracellular Matrix Degradation via the miR-149-5p/ HMGCR Axis.

BACKGROUND: Osteoarthritis (OA) is an inflammatory joint disorder with high incidence rates. Long non-coding RNAs (LncRNAs) influence OA development. OBJECTIVES: In this research, we attempt to figure out the functions of lncRNA BLACAT1 in human articular chondrocyte (HAC) apoptosis and extracellular matrix (ECM) degradation in OA. METHODS: Interleukin (IL)-1ß was employed to induce HAC damage. Cell viability and apoptosis were detected, with expression patterns of lncRNA BLACAT1, miR-149-5p, and HMGCR, and levels of Caspase-3, Caspase-9, BAX, Bcl-2, COL2A1, and SOX9 determined. Then, lncRNA BLACAT1 was silenced in IL-1ß-treated HACs to analyze its role in HAC damage. The target relations of lncRNA BLACAT1 and miR-149-5p and miR-149-5p and HMGCR were verified. In addition, combined experiments were performed as a miR-149-5p inhibitor or HMGCR overexpression was injected into cells with lncRNA BLACAT1 silencing. RESULTS: In IL-1ß-treated HACs, lncRNA BLACAT1 and HMGCR were overexpressed while miR- 149-5p was poorly expressed, along with reduced cell viability, enhanced apoptosis, elevated Caspase-3 and Caspase-9 activities, increased BAX level, decreased Bcl-2 level, and declined levels of COL2A1 and SOX9, which were reversed by lncRNA BLACAT1 silencing. LncRNA BLACAT1 targeted miR-149-5p, and miR-149-5p targeted HMGCR. miR-149-5p knockout or HMGCR overexpression annulled the inhibitory role of lncRNA BLACAT1 silencing in HAC apoptosis and ECM degradation. CONCLUSION: LncRNA BLACAT1 was overexpressed in IL-1ß-treated HACs, and the lncRNA BLACAT1/miR-149-5p/HMGCR ceRNA network promoted HAC apoptosis and ECM degradation.

Statin therapy inhibits fatty acid synthase via dynamic protein modifications.

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins might increase HMG-CoA levels and protein modifications. Upon statin treatment, we observe a strong increase in HMG-CoA levels and modification of only a single protein. Mass spectrometry identifies this protein as fatty acid synthase (FAS), which is modified on active site residues and, importantly, on non-lysine side-chains. The dynamic modifications occur only on a sub-pool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and ß-alanine metabolism.

Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and ß-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and ß-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing ß-caryophyllene synthase in SQ3-4 produced 11.86 ± 0.09 mg l-1 ß-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 ± 0.09 mg l-1 squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l-1 squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.