Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.
Ontology highlight
ABSTRACT:
SUBMITTER: Pojer F
PROVIDER: S-EPMC1544197 | biostudies-literature | 2006 Aug
REPOSITORIES: biostudies-literature
Publications
Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.
Pojer Florence F Ferrer Jean-Luc JL Richard Stéphane B SB Nagegowda Dinesh A DA Chye Mee-Len ML Bach Thomas J TJ Noel Joseph P JP
Proceedings of the National Academy of Sciences of the United States of America 20060724 31
3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic ana ...[more]
Similar Datasets
Project description:Simple Summary The cholesterol content in egg yolk is high, and an excessive consumption of egg yolk can have adverse effects on the health of the body. Therefore, it is necessary to systematically study the formation process of cholesterol in egg yolk and reduce the content of cholesterol in egg yolk. After using 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitors to inhibit the synthesis of cholesterol in the liver of laying hens, this study investigated the changes in the cholesterol content in egg yolk and the surrounding tissues and utilised RNA sequencing (RNA-seq) technology to explore the biological mechanism that is involved when HMGCR inhibitors reduce egg yolk cholesterol. Therefore, this study provides a reference for related research on cholesterol metabolism in laying hens. Abstract This study aimed to investigate the effect of HMGCR inhibitors on egg yolk cholesterol content and its biological mechanisms. Four groups of 180-day-old laying hens (n = 8 cages/group, 6 laying hens/cage) were fed a corn/soybean-based diet (control) and the control diet supplemented with an HMGCR inhibitor at 60, 150, and 300 mg/kg for 4 weeks. The experimental results showed that adding HMGCR inhibitors of 150 mg/kg or more can significantly reduce the cholesterol content in the liver, yolk, serum, and pectoral muscles of laying hens. The RNA-seq results showed that compared with the control group, the addition of HMGCR inhibitors of 150 mg/kg or more to the diet significantly upregulated genes related to cholesterol synthesis in the liver, and the genes involved in steroid synthesis and metabolism, sterol synthesis and metabolism, and cholesterol synthesis and metabolism were all affected by the HMGCR inhibitors. In summary, adding HMGCR inhibitors of 150 mg/kg or more to the diet of hens can significantly reduce the cholesterol content in egg yolk. After the HMGCR inhibitors inhibited the activity of the liver HMGCR, they also altered the expression of genes related to cholesterol synthesis, bile acid synthesis, and cholesterol transport in the liver, and ultimately reduced cholesterol synthesis and cholesterol transport to the egg yolk.
| S-EPMC10251945 | biostudies-literature
Project description:The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea. Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum. Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble. Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels.
| S-EPMC545772 | biostudies-literature
Project description:3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), the rate-limiting enzymes of sterol synthesis, undergoes feedback-regulated endoplasmic reticulum degradation in both mammals and yeast. The yeast Hmg2p isozyme is subject to ubiquitin-mediated endoplasmic reticulum degradation by the HRD pathway. We had previously shown that alterations in cellular levels of the 15-carbon sterol pathway intermediate farnesyl pyrophosphate (FPP) cause increased Hmg2p ubiquitination and degradation. We now present evidence that the FPP-derived, 20-carbon molecule geranylgeranyl pyrophosphate (GGPP) is a potent endogenous regulator of Hmg2p degradation. This work was launched by the unexpected observation that GGPP addition directly to living yeast cultures caused high potency and specific stimulation of Hmg2p degradation. This effect of GGPP was not recapitulated by FPP, GGOH, or related isoprenoids. GGPP-caused Hmg2p degradation met all the criteria for the previously characterized endogenous signal. The action of added GGPP did not require production of endogenous sterol molecules, indicating that it did not act by causing the build-up of an endogenous pathway signal. Manipulation of endogenous GGPP by several means showed that naturally made GGPP controls Hmg2p stability. Analysis of the action of GGPP indicated that the molecule works upstream of retrotranslocation and can directly alter the structure of Hmg2p. We propose that GGPP is the FPP-derived regulator of Hmg2p ubiquitination. Intriguingly, the sterol-dependent degradation of mammalian HMGR is similarly stimulated by the addition of GGOH to intact cells, implying that a dependence on 20-carbon geranylgeranyl signals may be a common conserved feature of HMGR regulation that may lead to highly specific therapeutic approaches for modulation of HMGR.
| S-EPMC2790966 | biostudies-literature
Project description:3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.
| S-EPMC6827278 | biostudies-literature
Project description:The formation of carbon-carbon bonds via an acyl-enzyme intermediate plays a central role in fatty acid, polyketide, and isoprenoid biosynthesis. Uniquely among condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) catalyzes the formation of a carbon-carbon bond by activating the methyl group of an acetylated cysteine. This reaction is essential in Gram-positive bacteria, and represents the first committed step in human cholesterol biosynthesis. Reaction kinetics, isotope exchange, and mass spectroscopy suggest surprisingly that HMGS is able to catalyze the "backwards" reaction in solution, where HMG-CoA is cleaved to form acetoacetyl-CoA (AcAc-CoA) and acetate. Here, we trap a complex of acetylated HMGS from Staphylococcus aureus and bound acetoacetyl-CoA by cryo-cooling enzyme crystals at three different times during the course of its back-reaction with its physiological product (HMG-CoA). This nonphysiological "backwards" reaction is used to understand the details of the physiological reaction with regards to individual residues involved in catalysis and substrate/product binding. The structures suggest that an active-site glutamic acid (Glu-79) acts as a general base both in the condensation between acetoacetyl-CoA and the acetylated enzyme, and the hydrolytic release of HMG-CoA from the enzyme. The ability to trap this enzyme-intermediate complex may suggest a role for protein dynamics and the interplay between protomers during the normal course of catalysis.
| S-EPMC534525 | biostudies-literature
Project description:The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC in vitro and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells in vitro. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.
| S-EPMC4094470 | biostudies-literature
Project description:We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass. Identify of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase. (ii) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase. (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity. (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme. The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria. These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme.
| S-EPMC54006 | biostudies-other
Project description:BACKGROUND: 3-hydroxy-3-methylglutaric aciduria (3HMG, McKusick: 246450) is an autosomal recessive branched chain organic aciduria caused by deficiency of the enzyme 3-Hydroxy-3-Methylglutaryl CoA lyase (HL, HMGCL, EC 4.1.3.4). HL is encoded by HMGCL gene and many mutations have been reported. 3HMG is commonly observed in Saudi Arabia. METHODS: We utilized Whole Genome Amplification (WGA), PCR and direct sequencing to identify mutations underlying 3HMG in the Saudi population. Two patients from two unrelated families and thirty-four 3HMG positive dried blood spots (DBS) were included. RESULTS: We detected the common missense mutation R41Q in 89% of the tested alleles (64 alleles). 2 alleles carried the frame shift mutation F305fs (-2) and the last two alleles had a novel splice site donor IVS6+1G>A mutation which was confirmed by its absence in more than 100 chromosomes from the normal population. All mutations were present in a homozygous state, reflecting extensive consanguinity. The high frequency of R41Q is consistent with a founder effect. Together the three mutations described account for >94% of the pathogenic mutations underlying 3HMG in Saudi Arabia. CONCLUSION: Our study provides the most extensive genotype analysis on 3HMG patients from Saudi Arabia. Our findings have direct implications on rapid molecular diagnosis, prenatal and pre-implantation diagnosis and population based prevention programs directed towards 3HMG.
| S-EPMC1764877 | biostudies-literature
Project description:3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.
| S-EPMC3460428 | biostudies-literature
Project description:3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD) is a rare autosomal recessively inherited metabolic disorder. Patients suffer from avoidable neurologically devastating metabolic decompensations and thus would benefit from newborn screening (NBS). The diagnosis is currently made by measuring dry blood spot acylcarnitines (C5OH and C6DC) followed by urinary organic acid profiling for the differential diagnosis from several other disorders. Using untargeted metabolomics (reversed-phase UHPLC coupled to an Orbitrap Elite hybrid mass spectrometer) of plasma samples from 5 HMGCLD patients and 19 age-matched controls, we found 3-methylglutaconic acid and 3-hydroxy-3-methylglutaric acid, together with 3-hydroxyisovalerylcarnitine as the most discriminating metabolites between the groups. In order to evaluate the NBS potential of these metabolites we quantified the most discriminating metabolites from untargeted metabolomics in 23 blood spots from 4 HMGCLD patients and 55 controls by UHPLC tandem mass spectrometry. The results provide a tool for expanded NBS of HMGCLD using tandem mass spectrometry. Selected reaction monitoring transition 262/85 could be used in a first-tier NBS analysis to screen for elevated 3-hydroxyisovalerylcarnitine. In a positive case, a second-tier analysis of 3-hydroxy-3-methylglutaric acid and 3-methylglutaconic acid in a dry blood spot using UHPLC tandem mass spectrometry instruments confirms the diagnosis. In conclusion, we describe the identification of new diagnostic biomarkers for HMGCLD and their application in NBS in dry blood spots. By using second-tier testing, all patients with HMGCLD were unequivocally and correctly diagnosed.
| S-EPMC7358667 | biostudies-literature