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Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene.

ABSTRACT: Acute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcription factors (SREBP-1, SREBP-2, NF-kappaB, c-Fos, and c-Jun), and heat shock proteins (HSP-70 and heme oxygenase-1) to the HMGCR promoter and transcription region (start/end exons) were assessed by Matrix ChIP assay. HMGCR mRNA, protein, and cholesterol levels were determined. Finally, histone modifications at HMGCR were assessed. Ischemia/reperfusion (I/R) induced marked cholesterol loading, which corresponded with elevated Pol II recruitment to HMGCR and increased expression levels of both HMGCR protein and mRNA. I/R also induced the binding of multiple transcription factors (SREBP-1, SREBP-2, c-Fos, c-Jun, NF-kappaB) and heat shock proteins to the HMGCR promoter and transcription regions. Significant histone modifications (increased H3K4m3, H3K19Ac, and H2A.Z variant) at these loci were also observed but were not identified at either the 5' and 3' HMGCR flanking regions (+/-5000 bps) or at negative control genes (beta-actin and beta-globin). In conclusion, I/R activates the HMGCR gene via multiple stress-activated transcriptional and epigenetic pathways, contributing to renal cholesterol loading.

SUBMITTER: Naito M

PROVIDER: S-EPMC2631318 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene.

Naito Masayo M Bomsztyk Karol K Zager Richard A RA

The American journal of pathology 20081218 1

Acute kidney injury evokes renal tubular cholesterol synthesis. However, the factors during acute kidney injury that regulate HMG CoA reductase (HMGCR) activity, the rate-limiting step in cholesterol synthesis, have not been defined. To investigate these factors, mice were subjected to 30 minutes of either unilateral renal ischemia or sham surgery. After 3 days, bilateral nephrectomy was performed and cortical tissue extracts were prepared. The recruitment of RNA polymerase II (Pol II), transcri ...[more]

PMID: 19095962

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Project description:BackgroundHypercholesterolemia has posed a serious threat of heart diseases and stroke worldwide. Xanthine oxidase (XO), the rate-limiting enzyme in uric acid biosynthesis, is regarded as the root of reactive oxygen species (ROS) that generate atherosclerosis and cholesterol crystals. β-Hydroxy β-methylglutaryl-coenzyme A reductase (HMGR) is a rate-limiting enzyme in cholesterol biosynthesis. Although some commercially available enzyme inhibiting drugs have effectively reduced cholesterol levels, most of them have failed to meet potential drug candidates' requirements. Here, we have carried out an in-silico analysis of secondary metabolites that have already shown good inhibitory activity against XO and HMGR in a wet lab setup.MethodsOut of 118 secondary metabolites reviewed, sixteen molecules inhibiting XO and HMGR were selected based on the IC50 values reported in in vitro assays. Further, receptor-based virtual screening was carried out against secondary metabolites using GOLD Protein-Ligand Docking Software, combined with subsequent post-docking, to study the binding affinities of ligands to the enzymes. In-silico ADMET analysis was carried out to explore their pharmacokinetic properties, followed by toxicity prediction through ProTox-II.ResultsThe molecular docking of amentoflavone (GOLD score 70.54, ∆G calc. = - 10.4 Kcal/mol) and ganomycin I (GOLD score 59.61, ∆G calc. = - 6.8 Kcal/mol) displayed that the drug has effectively bound at the competitive site of XO and HMGR, respectively. Besides, 6-paradol and selgin could be potential drug candidates inhibiting XO. Likewise, n-octadecanyl-O-α-D-glucopyranosyl (6' → 1″)-O-α-D-glucopyranoside could be potential drug candidates to maintain serum cholesterol. In-silico ADMET analysis has shown that these sixteen metabolites were optimal within the categorical range compared to commercially available XO and HMGR inhibitors, respectively. Toxicity analysis through ProTox-II revealed that 6-gingerol, ganoleucoin K, and ganoleucoin Z are toxic for human use.ConclusionThis computational analysis supports earlier experimental evidence towards the inhibition of XO and HMGR by natural products. Further study is necessary to explore the clinical efficacy of these secondary molecules, which might be alternatives for the treatment of hypercholesterolemia.

Project description:HMG-CoA reductase (HMGR) is the target of statins, cholesterol-lowering drugs prescribed to millions of patients worldwide. More recent research indicates that HMGR could be a useful target in the development of antimicrobial agents. Over the last seven decades, researchers have proposed a series of increasingly complex reaction mechanisms for this biomedically important enzyme. The maturation of the mechanistic proposals for HMGR have paralleled advances in a diverse set of research areas, such as molecular biology and computational chemistry. Thus, the development of the HMGR mechanism provides a useful case study for following the advances in state-of-the-art methods in enzyme mechanism research. Similarly, the questions raised by these mechanism proposals reflect the limitations of the methods used to develop them. The mechanism of HMGR, a four-electron oxidoreductase, is unique and far more complex than originally thought. The reaction contains multiple chemical steps, coupled to large-scale domain motions of the homodimeric enzyme. The first proposals for the HMGR mechanism were based on kinetic and labeling experiments, drawing analogies to the mechanism of known dehydrogenases. Advances in molecular biology and bioinformatics enabled researchers to use site-directed mutagenesis experiments and protein sequencing to identify catalytically important glutamate, aspartate, and histidine residues. These studies, in turn, have generated new and more complicated mechanistic proposals. With the development of protein crystallography, researchers solved HMGR crystal structures to reveal an unexpected lysine residue at the center of the active site. The many crystal structures of HMGR led to increasingly complex mechanistic proposals, but the inherent limitations of the protein crystallography left a number of questions unresolved. For example, the protonation state of the glutamate residue within the active site cannot be clearly determined from the crystal structure. The differing protonation state of this residue leads to different proposed mechanisms for the enzyme. As computational analysis of large biomolecules has become more feasible, the application of methods such as hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to the HMGR mechanism have led to the most detailed mechanistic proposal yet. As these methodologies continue to improve, they prove to be very powerful for the study of enzyme mechanisms in conjunction with protein crystallography. Nevertheless, even the most current mechanistic proposal for HMGR remains incomplete due to limitations of the current computational methodologies. Thus, HMGR serves as a model for how the combination of increasingly sophisticated experimental and computational methods can elucidate very complex enzyme mechanisms.

Dataset Information

Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene.

Publications

Renal ischemia-induced cholesterol loading: transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene.

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