The cloning and molecular regulation of rat 3-hydroxy-3-methylglutaryl coenzyme A reductase
Williams, David Lee
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Permalink
https://hdl.handle.net/2142/23293
Description
Title
The cloning and molecular regulation of rat 3-hydroxy-3-methylglutaryl coenzyme A reductase
Author(s)
Williams, David Lee
Issue Date
1990
Doctoral Committee Chair(s)
Shapiro, David J.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
We have cloned and sequenced the cDNA for rat HMG-CoA reductase the key regulatory enzyme in the mevalonate pathway. The amino acid sequence of rat HMG-CoA reductase is highly homologous to hamster and human reductases. Noncoding regions of the reductase mRNAs are also highly conserved. Rat HMG-CoA reductase mRNA is initiated at a single cluster of start sites about 105 bases from the start of translation. Six conserved serines in the linker domain has been identified as possible sites for protein phosphorylation.
To identify a peroxisomal targeting sequence in the carboxy terminus of rat HMG-CoA reductase fusions of chloramphenicol acetyltransferase (CAT) and the 30 carboxyterminal amino acids of reductase were expressed in cultured cells. The subcellular localization of expressed proteins was determined using indirect immunofluorescence microscopy. CAT protein in control transfections was found in the cytoplasm and nuclei. No CAT-reductase fusion protein was detected. Therefore we were unable to locate a peroxisomal targeting sequence in rat HMG-CoA reductase.
An oxidosqualene cyclase inhibitor was used to block sterol synthesis and maximize endproduct feedback from the nonsterol arm of the mevalonate pathway. The inhibitor caused a rapid decline in both reductase activity and mRNA levels. The mechanism of action of the inhibitor is the same as sterols, repressing HMG-CoA reductase gene transcription.
The effect of hormones on rat HMG-CoA reductase mRNA levels was investigated. Dexamethasone caused a transient 2-3 fold increase in reductase mRNA levels while insulin and thyroid hormone had no effect. The half life of rat HMG-CoA reductase mRNA was measured directly for the first time and determined to be 4.5 $\pm$ 0.4 hours. Dexamethasone increases the level of reductase mRNA by increasing the reductase gene transcription rate.
These results indicate that rat reductase gene transcription is regulated by a single mechanism. This suggests that HMG-CoA reductase has a much more simple transcriptional control mechanism than other genes studied. We propose that the HMG-CoA reductase promoter contains a single regulatory element which is responsive to sterols. The gene for HMG-CoA reductase would be constitutively active with sterols repressing this activity.
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