Abstract
Holocarboxylase synthetase (HCS) catalyzes the biotinylation of five carboxylases in human cells, and mutations of HCS cause multiple carboxylase deficiency (MCD). Although HCS also participates in the regulation of its own mRNA levels, the relevance of this mechanism to normal metabolism or to the MCD phenotype is not known. In this study, we show that mRNA levels of enzymes involved in biotin utilization, including HCS, are down-regulated during biotin deficiency in liver while remaining constitutively expressed in brain. We propose that this mechanism of regulation is aimed at sparing the essential function of biotin in the brain at the expense of organs such as liver and kidney during biotin deprivation. In MCD, it is possible that some of the manifestations of the disease may be associated with down-regulation of biotin utilization in liver because of the impaired activity of HCS and that high dose biotin therapy may in part be important to overcoming the adverse regulatory impact in such organs.
Highlights
The vitamin biotin functions as the cofactor in carboxylation reactions where it acts as the carboxyl carrier
Biotin Availability Regulates mRNA Levels of the Biotin Utilization Cycle Proteins in HepG2 Cells—To determine whether key components of the biotin cycle are under the control of the exogenous biotin concentration, we examined mRNA levels of sodiumdependent multivitamin transporter (SMVT) and biotinidase, two proteins that control entry of biotin into the cycle
We assessed pyruvate carboxylase (PC) mRNA because of its central role in metabolism and compared all three with Holocarboxylase synthetase (HCS), which we showed previously to be regulated by biotin availability [29]
Summary
Materials—Biotin and 8-bromo-cyclic guanosine monophosphate (8Br-cGMP) were from Sigma-Aldrich. PCR products were evaluated following electrophoresis on agarose gels and were quantified by densitometry using a molecular imager FX (Bio-Rad) and normalized to -actin mRNA as described previously [29]. For Western blot analysis, crude extracts from human cells or rat tissues were prepared in radioimmune precipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 5 l of protease inhibitor mixture (Roche Applied Science) and 0.5 l of 200 mM phenylmethylsulfonyl fluoride (Invitrogen). Total RNA was isolated from brain, liver, and kidney from normal and biotindeficient rats, and mRNA levels were determined as described above. Results of biotin starvation on mRNA were normalized to -actin mRNA and expressed as a percentage of mRNA levels observed in cells or rats grown in biotin-replete medium. Data are presented as the mean of three different experiments Ϯ S.E
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