Characterization of Peroxisomal 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase in UT2* Cells:  Sterol Biosynthesis, Phosphorylation, Degradation, and Statin Inhibition

Aboushadi, Nahla; Shackelford, Janis E.; Jessani, Nadim; Gentile, and Angela; Krisans, Skaidrite K.

doi:10.1021/bi9916325

Cited by 22 publications

(14 citation statements)

References 53 publications

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“…33 Despite these mutations, the UT2* cell line expresses a smaller but enzymatically active isoform of HMGCR that has lower specific activity (52 versus 275 pmol·min Ϫ1 ·mg Ϫ1 ) and statin sensitivity compared with the normal isoform. 34 The authors were unable to identify an HMGCR transcript that could account for this novel isoform; however, this finding suggests that splicing mutations around exon 13 can result in an active, and partially statin-resistant, isoform.…”

Section: Discussionmentioning

confidence: 95%

Alternative Splicing of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Is Associated With Plasma Low-Density Lipoprotein Cholesterol Response to Simvastatin

Medina¹,

et al. 2008

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Background-HMGCR (3-Hydroxy-3-methylglutaryl coenzyme A reductase), the direct target of statin inhibition, undergoes alternative splicing of exon 13, which encodes part of the statin-binding domain of the enzyme. We hypothesized that HMGCR alternative splicing might be related to the interindividual variation in plasma low-density lipoprotein cholesterol response to statin treatment. Methods and Results-We measured mRNA expression of both the full-length and the alternatively spliced HMGCR transcript lacking exon 13 (HMGCRv_1) in 170 simvastatin-incubated immortalized lymphocyte cell lines derived from participants in the Cholesterol and Pharmacogenetics (CAP) study who were treated with simvastatin 40 mg/d for 6 weeks. Greater upregulation of HMGCRv_1 in vitro was significantly correlated (PՅ0.0001) with smaller in vivo reductions of plasma total cholesterol, low-density lipoprotein cholesterol, apoprotein B, and triglycerides and explained 6% to 15% of the variation in their response to treatment. In contrast, no significant relationship was found between expression of the full-length HMGCR transcript and in vivo response. By siRNA knockdown of the full-length transcript, we found that HMGCR enzyme activity measured in cells enriched in HMGCRv_1 was relatively resistant to statin inhibition, consistent with the association of increased alternative splicing with reduced statin response in the CAP study. In addition, we found that a common HMGCR single-nucleotide polymorphism (rs3846662) located within intron 13 was associated with variation in the proportion of HMGCR mRNA that is alternatively spliced. Conclusions-Variation in the production of an HMGCR isoform with reduced statin sensitivity is a determinant of interindividual differences in low-density lipoprotein cholesterol, apolipoprotein B, and triglyceride response to statin treatment. (Circulation. 2008;118:355-362.)

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Section: Discussionmentioning

confidence: 95%

Alternative Splicing of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Is Associated With Plasma Low-Density Lipoprotein Cholesterol Response to Simvastatin

Medina¹,

et al. 2008

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“…Recently, Engfelt et al (16) identified the responsible mutations in the structural gene for reductase. By growing UT-2 cells in the absence of mevalonate, they have established a new cell line, designated as UT-2* cells, and demonstrated that UT-2* cells had up-regulated HMGCoA reductase activity, which was localized exclusively to peroxisomes and the peroxisomal enzyme in these cells was sufficient for survival without mevalonate (15,17). Based on the detailed characterization of UT-2* cells, they suggested the existence of a second, peroxisome-specific gene for the reductase.…”

Section: Discussionmentioning

confidence: 99%

“…Given the major role of HMG-CoA reductase in the mevalonate pathway, it is tempting to hypothesize that mammalian cells also have a second gene for the enzyme. In fact, although the classic form of the enzyme is a transmembrane protein anchored to the endoplasmic reticulum (ER), recent studies using a mutant cell line that lacks the ER isoform of the enzyme indicate the existence of a second isoform of the reductase exclusively localized in peroxisomes and that the peroxisomal activity might be due to a second gene (15)(16)(17).…”

Section: -3)mentioning

confidence: 99%

Early Embryonic Lethality Caused by Targeted Disruption of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene

Ohashi

Osuga

Tozawa

et al. 2003

Journal of Biological Chemistry

103

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The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr؉/؊) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr؉/؊ embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr؉/؊ mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr؉/؊ and Hmgcr؉/؉ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.The mevalonate pathway produces isoprenoids that are essential for diverse cellular functions, ranging from cholesterol synthesis to growth control. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) 1 reductase (EC 1.1.1.34), which catalyzes the conversion of HMG-CoA to mevalonate, is the rate-limiting enzyme in the mevalonate pathway (1). Because of its major role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely studied. To ensure a steady mevalonate supply, the non-sterol and sterol end-products of mevalonate metabolism exert feedback regulation on the activity of this enzyme through multivalent mechanisms, including inhibition of transcription of the HMG-CoA reductase mRNA, blocking of translation, and acceleration of protein degradation, thus regulating the amount of reductase protein over a several hundred-fold range (reviewed in Refs.

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“…The activity in peroxisomes was less than 5% of the total HMGCR in the cell under control conditions, but reached up to 30% after treatment with cholestyramine (11). The presence of significant amounts of peroxisomal HMGCR has been confirmed by other groups (12,13) and in other tissues and organisms [hamster ovarian epithelium (CHO cells) (14)(15)(16); rat brain (17)]. …”

Section: Subcellular Distribution Of Classical Hmg-coa Reductasementioning

confidence: 79%

“…How does mevalonate withdrawal cause a stable [inheritable? see (16)] change in HMGCR expression? Do the cloned transcripts really represent the complete diversity of existing transcripts, given that so many different splice variants are observed in UT2* cells, although the mutations are localized and well defined?…”

Section: Subcellular Distribution Of Classical Hmg-coa Reductasementioning

confidence: 99%

A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment

Breitling

Krisans

2002

Journal of Lipid Research

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HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step of eukaryotic isoprenoid biosynthesis, and is the main target of cholesterol-lowering drugs. The classical form of the enzyme is a transmembrane-protein anchored to the endoplasmic reticulum. However, during the last years several lines of evidence pointed to the existence of a second isoform of HMGCR localized in peroxisomes, where mevalonate is converted further to farnesyl diphosphate. This finding is relevant for our understanding of the complex regulation and compartmentalization of the cholesterogenic pathway. Here we review experimental evidence suggesting that the peroxisomal activity might be due to a second HMGCR gene in mammals. We then present a comprehensive analysis of completely sequenced eukaryotic genomes, as well as the human and mouse genome drafts. Our results provide evidence for a large number of independent duplications of HMGCR in all eukaryotic kingdoms, but not for a second gene in mammals. We conclude that the peroxisomal HMGCR activity in mammals is due to alternative targeting of the ER enzyme to peroxisomes by an as yet uncharacterized mechanism. -Breitling, R., and S. K. Krisans. A second gene for peroxisomal HMGCoA reductase? A genomic reassessment.

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Characterization of Peroxisomal 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase in UT2* Cells: Sterol Biosynthesis, Phosphorylation, Degradation, and Statin Inhibition

Cited by 22 publications

References 53 publications

Alternative Splicing of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Is Associated With Plasma Low-Density Lipoprotein Cholesterol Response to Simvastatin

Alternative Splicing of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Is Associated With Plasma Low-Density Lipoprotein Cholesterol Response to Simvastatin

Early Embryonic Lethality Caused by Targeted Disruption of the 3-Hydroxy-3-methylglutaryl-CoA Reductase Gene

A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment

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