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• W2110134612 abstract "Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5′-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation. Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5′-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation. In humans, the vitamin biotin is an essential micronutrient that has two different functions in the cell (1Pacheco-Alvarez D. Solórzano-Vargas R.S. Leon-Del-Rio A. Arch. Med. Res. 2002; 33: 439-447Crossref PubMed Scopus (99) Google Scholar). First, it is the cofactor of five biotin-dependent carboxylases: pyruvate carboxylase (PC), 3The abbreviations used are: PC, pyruvate carboxylase; HCS, holocarboxylase synthetase; MCD, multiple carboxylase deficiency; B-AMP, 5′-biotinyl-AMP; PCC, propionyl-CoA-carboxylase; ACC, acetyl-CoA carboxylase; MCC, methylcrontonyl-CoA carboxylase; sGC, soluble guanylate cyclase; PKG, cGMP-dependent protein kinase; ODQ, 1-H(1,2,4)-oxadiazolo-[4,3-a]quinaxolin-1-one; 8-Br-cGMP, 8-bromoguanosine 3′,5′-cyclic monophosphate; PVDF, polyvinylidene difluoride. 3The abbreviations used are: PC, pyruvate carboxylase; HCS, holocarboxylase synthetase; MCD, multiple carboxylase deficiency; B-AMP, 5′-biotinyl-AMP; PCC, propionyl-CoA-carboxylase; ACC, acetyl-CoA carboxylase; MCC, methylcrontonyl-CoA carboxylase; sGC, soluble guanylate cyclase; PKG, cGMP-dependent protein kinase; ODQ, 1-H(1,2,4)-oxadiazolo-[4,3-a]quinaxolin-1-one; 8-Br-cGMP, 8-bromoguanosine 3′,5′-cyclic monophosphate; PVDF, polyvinylidene difluoride. propionyl-CoA carboxylase (PCC), methylcrotonyl-CoA carboxylase (MCC), and two forms of acetyl-CoA carboxylase (ACC-1 and ACC-2) (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). These enzymes catalyze key reactions in gluconeogenesis, branched chain amino acid catabolism, and fatty acid synthesis and underscore the importance of biotin to metabolic homeostasis (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). Second, biotin is a regulator of the expression of several hepatic proteins that include glucokinase, phosphoenol pyruvate carboxykinase, and most of the proteins involved in biotin metabolism (1Pacheco-Alvarez D. Solórzano-Vargas R.S. Leon-Del-Rio A. Arch. Med. Res. 2002; 33: 439-447Crossref PubMed Scopus (99) Google Scholar, 3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar, 5Pacheco-Alvarez D. Solórzano-Vargas R.S. González-Noriega A. Michalak C. Zempleni J. León-Del-Río A. Mol. Genet. Metab. 2005; 85: 301-307Crossref PubMed Scopus (31) Google Scholar, 6Chauhan J. Dakshinamurti K. J. Biol. Chem. 1991; 266: 10035-10038Abstract Full Text PDF PubMed Google Scholar, 7Collins J. Paietta E. Green R. Morell A. Stockert R. J. Biol. Chem. 1988; 263: 11280-11283Abstract Full Text PDF PubMed Google Scholar, 8Deodhar A.D. Mistry S.P. Life Sci. II. 1970; 9: 581-588Crossref PubMed Scopus (22) Google Scholar, 9Dakshinamurti K. Tarrago-Litvak L. Hong H.C. Can. J. Biochem. 1970; 48: 493-500Crossref PubMed Scopus (34) Google Scholar, 10Borboni P. Magnaterra R. Rabini R.A. Staffolani R. Porzio O. Sesti G. Fusco A. Mazzanti L. Lauro R. Marlier L.N. Acta Diabetol. 1996; 33: 154-158Crossref PubMed Scopus (51) Google Scholar, 11De la Vega L.A. Stockert R.J. Am. J. Physiol. 2000; 279: C2037-C2042Crossref PubMed Google Scholar, 12Maeda Y. Kawata S. Inui Y. Fukuda K. Igura T. Matsuzawa Y. J. Nutr. 1996; 126: 61-66Crossref PubMed Scopus (26) Google Scholar, 13Rodriguez-Melendez R. Cano S. Mendez S.T. Velazquez A. J. Nutr. 2001; 131: 1909-1913Crossref PubMed Scopus (75) Google Scholar). Because of the importance of biotin in cell metabolism, higher organisms face a constant threat to their survival because they are incapable of synthesizing the vitamin. The situation is further complicated by the limited availability of biotin in nature, most of which is protein-bound and not directly accessible (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar, 14Pispa J. Ann. Med. Exp. Biol. Fenn. 1965; 43: 1-39PubMed Google Scholar). During evolution, mammals developed what is known as the biotin cycle, which allows them to cope with the low availability of this critical nutrient (Fig. 1). This system depends on two enzymes; holocarboxylase synthetase (HCS) and biotinidase (1Pacheco-Alvarez D. Solórzano-Vargas R.S. Leon-Del-Rio A. Arch. Med. Res. 2002; 33: 439-447Crossref PubMed Scopus (99) Google Scholar, 2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). HCS is responsible for the activation, via biotinylation, of all biotin-dependent carboxylases in human cells. The process takes place in a two-step, ATP-dependent reaction in which biotin is first activated to 5′-biotinyl-AMP (B-AMP) and then transferred to a specific and highly conserved lysine residue in all biotin-dependent carboxylases (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar, 15Chapman-Smith A. Cronan Jr., J.E. Trends Biochem. Sci. 1999; 24: 359-363Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Leon-Del-Rio A. Gravel R.A. J. Biol. Chem. 1994; 269: 22964-22968Abstract Full Text PDF PubMed Google Scholar). Biotinidase catalyzes the release of biotin from biotinylated peptides or biocytin (biotinyl-lysine), products generated by intestinal digestion of nutrient proteins or during carboxylase turnover (endogenous biotin recycling) (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar, 17Cole H. Reynolds T.R. Lockyer J.M. Buck G.A. Denson T. Spence J.E. Hymes J. Wolf B. J. Biol. Chem. 1994; 269: 6566-6570Abstract Full Text PDF PubMed Google Scholar). We recently showed that HCS is an obligate participant in biotin-mediated transcriptional regulation (Fig. 1). The underlying mechanism requires B-AMP, the product of the HCS reaction, which activates a signal transduction cascade involving soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) (4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar, 7Collins J. Paietta E. Green R. Morell A. Stockert R. J. Biol. Chem. 1988; 263: 11280-11283Abstract Full Text PDF PubMed Google Scholar). In the presence of biotin, the HCS-sGC-PKG pathway induces the expression of the components of the biotin cycle required for its transport and utilization: the sodium-dependent multivitamin transporter, PC, PCC, MCC, ACC, and HCS (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 5Pacheco-Alvarez D. Solórzano-Vargas R.S. González-Noriega A. Michalak C. Zempleni J. León-Del-Río A. Mol. Genet. Metab. 2005; 85: 301-307Crossref PubMed Scopus (31) Google Scholar, 13Rodriguez-Melendez R. Cano S. Mendez S.T. Velazquez A. J. Nutr. 2001; 131: 1909-1913Crossref PubMed Scopus (75) Google Scholar). Paradoxically, biotin deficiency results in reduced expression of these genes in tissues such as liver, kidney, and muscle but not brain. Although this would seem to be contrary to the need for scavenging biotin during limited supply, we showed that this pattern of gene repression is an altruistic tissue-specific contingency mechanism that, by down-regulating biotin utilization in selected tissues, allows a concerted supply of the remaining vitamin to the brain (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In this organ, PC plays two essential roles: as a key player in anaplerosis of the Krebs cycle through pyruvate carboxylation and in the restoration of ;-ketoglutarate lost during the release of glutamate and ;-aminobutyric acid from neurons and glutamine export from glia (18Chiang G. Mistry S. Proc. Soc. Exp. Biol. Med. 1974; 146: 21-24Crossref PubMed Scopus (27) Google Scholar, 19Hassel B. Mol. Neurobiol. 2000; 22: 21-40Crossref PubMed Google Scholar). In humans, the biotin cycle can be disrupted by genetic deficiency of holocarboxylase synthetase (HCS deficiency (MIM 253270)) or biotinidase (BTD deficiency (MIM 253260)), resulting in neonatal or juvenile onset forms, respectively, of the disease multiple carboxylase deficiency (MCD) (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar, 20Wolf B. Grier R. Secor McVoy J.R. Heard G. J. Inherit. Metab. Dis. 1985; 8: 53-58Crossref PubMed Scopus (125) Google Scholar, 21Dupuis L. Campeau E. Leclerc D. Gravel R.A. Mol. Genet. Metab. 1999; 66: 80-90Crossref PubMed Scopus (37) Google Scholar). Although the two diseases differ in the age of onset of symptoms, they share a number of clinical and biochemical manifestations, including decreased activities of all carboxylases, organic acidemia, hyperammonemia, dermatitis, alopecia, seizures, and coma. In biotinidase-deficient patients, neurological damage may also include mental retardation, hearing loss, and optic nerve atrophy (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). Although potentially lethal, most of clinical and biochemical manifestations of neonatal and juvenile MCD can be successfully treated with pharmacological doses of biotin. The biotin-responsiveness of neonatal MCD patients is associated primarily with having at least one allele expressing a mutant HCS with an elevated Km for biotin, which allows for increased functional activity at high concentrations of biotin (1Pacheco-Alvarez D. Solórzano-Vargas R.S. Leon-Del-Rio A. Arch. Med. Res. 2002; 33: 439-447Crossref PubMed Scopus (99) Google Scholar, 2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar, 3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar, 5Pacheco-Alvarez D. Solórzano-Vargas R.S. González-Noriega A. Michalak C. Zempleni J. León-Del-Río A. Mol. Genet. Metab. 2005; 85: 301-307Crossref PubMed Scopus (31) Google Scholar). However, based on the participation of HCS in the biotin-dependent transcriptional regulation of the biotin cycle, we have suggested that the clinical and biochemical deficits in HCS-deficient patients reflect the combined effects of the low affinity of the mutant enzyme for biotin and the concomitant reduction in carboxylase and HCS mRNA levels (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar). In biotinidase-deficient patients, the biotin cycle is largely intact because free biotin can be successfully utilized for the biotinylation of carboxylases. Here the deficit has been thought to be in the inadequacy of the biotin supply because of the inability to recycle protein-bound biotin from endogenous or nutrient sources (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). Therefore, supplementation with biotin at pharmacologic doses is thought to compensate for the loss of access to the additional biotin that would normally be available from protein sources (2Wolf B. Scriver C. Beaudet A.L. William S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Medical Publishing Division, New York2001: 3935-3962Google Scholar). Although the ultimate consequence of biotinidase deficiency is the interruption of the metabolic pathways where biotin-dependent carboxylases participate, the clinical manifestations that distinguish this disorder from HCS deficiency, especially in relation to neurological presentation, are not fully understood (22Sander J.E. Packman S. Townsend J.J. Neurology. 1982; 32: 878-880Crossref PubMed Google Scholar). In this work, we use fibroblasts from a biotinidase-deficient patient as an experimental model to study the role of this enzyme in carboxylase biotinylation and in HCS-sGC-PKG-dependent expression of biotin-dependent carboxylases and HCS under conditions of biotin deficiency and supplementation. Our results show that in biotinidase-deficient cells, biotin starvation results in a more rapid reduction in carboxylase biotinylation and in the expression of PC and MCC than in normal fibroblasts. We also demonstrate that in biotinidase-deficient cells the expression and activity of HCS is lower than in control cells. We propose that, in the absence of biotin supplementation, biotinidase-deficient patients may develop a secondary HCS deficiency that, combined with the primary biotinidase deficiency, may disrupt the altruistic regulation of biotin utilization that protects brain metabolism against vitamin starvation. Materials—Biotin, biocytin, 1-H(1,2,4)-oxadiazolo-[4,3-a]quinaxolin-1-one (ODQ), and 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) were from Sigma-Aldrich. d-[8,9–3H]biotin (34.0 Ci/mmol) was purchased to Amersham Biosciences. The biotinidase-deficient cell line MCD-BD was from the Montreal Children's Hospital Cell Repository for Mutant Human Cell Strains, and the HCS-deficient cell line MCD-MK (23Dupuis L. Leon-Del-Rio A. Leclerc D. Campeau E. Sweetman L. Saudubray J.M. Herman G. Gibson K.M. Gravel R.A. Hum. Mol. Genet. 1996; 5: 1011-1016Crossref PubMed Scopus (51) Google Scholar) and the rabbit antibody to HCS (24Narang M.A. Dumas R. Ayer L.M. Gravel R.A. Hum. Mol. Genet. 2004; 13: 15-23Crossref PubMed Scopus (114) Google Scholar) were reported previously. Human normal fibroblasts were obtained from G. Soldevila (Universidad Nacional Autónoma de Méx-ico). Escherichia coli C-124 cells have been previously described (16Leon-Del-Rio A. Gravel R.A. J. Biol. Chem. 1994; 269: 22964-22968Abstract Full Text PDF PubMed Google Scholar, 25Leon-Del-Rio A. Leclerc D. Akerman B. Wakamatsu N. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4626-4630Crossref PubMed Scopus (90) Google Scholar). Cell cultures were maintained in ;-minimum Eagle's medium containing high glucose (Invitrogen; biotin concentration, 0.40 ;m) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 ;g/ml streptomycin (biotin-replete medium). Biotin-deficient medium was prepared using biotin-free minimum Eagle's medium, dialyzed fetal bovine serum (Invitrogen), and the same antibiotic concentration. Cell Culture Experiments—The methods for biotin starvation of cell cultures are essentially as described previously (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar). Briefly, the cells were grown in biotin-replete or biotin-deficient medium at 37 °C with 5% CO2 for up to 13 days. The medium was changed at 3-day intervals. For carboxylase biotinylation recovery experiments, the cells grown in biotin-deficient medium were stimulated with biotin or biocytin, at concentrations from 1 to 100 nm for 24 h. Western Blot Analysis for HCS Expression and Carboxylase Biotin Content—Crude extracts from human cell cultures (30–100 ;g of total protein) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were incubated in a 1:3000 solution of rabbit HCS antibody or with a 1:2000 solution of streptavidin-AP conjugate (Roche Applied Science) or with a 1:500 solution of goat MCC or PCC antibodies (Santa Cruz Biotechnology). Visualization of HCS bands was performed using a BM chemiluminescence Western blotting kit (Roche Applied Science). Biotin-containing bands were quantitated using an FX image analyzer (Bio-Rad) as described above. The protein concentration used in these experiments was determined using a ND-1000 spectrophotometer (Nanodrop Technologies, Inc.), and confirmation of equal amounts of total protein in every lane was done by staining the gels with Coomassie Blue before protein transfer to PVDF membranes. Effect of Biotin and cGMP on Carboxylase Expression in Biotin-starved Cells—To determine the involvement of sGC on the recovery of biotinylation and carboxylase protein levels, biotin-starved normal fibroblasts were treated with 50 ;m ODQ, a specific inhibitor of sGC, for 3 h (4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar). After this period, 1 ;m biotin was added to the medium for 48 or 72 h, and the effect on MCC protein levels was compared with biotin-deficient cells stimulated by biotin without ODQ and cells grown continuously in normal medium (control cells). Alternatively, MCD-BD cells grown in biotin-free medium for 13 days were stimulated with 1 ;m biotin or 1 mm 8-Br-cGMP, a nonhydrolyzable analogue of cGMP. The cells were harvested after 48 or 72 h, and the MCC protein levels were determined as described above. Reverse Transcription-PCR—Procedures for RNA isolation, cDNA synthesis, and PCR have been previously described (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar). 5 ;g of total RNA and 0.5 ;m gene-specific oligonucleotide primers were used for cDNA synthesis and 0.3 ;m specific sense and antisense primers were used to give 200–300 bp of PCR products. The oligonucleotides used to amplify human mRNA were: HCS: 5′-CCC GAG CTC CGT CTC CTG GAT CGG-3′ and 5′-CCC AAG CCT TTT ACC GCC GTT TGG GGA-3′ (Tm = 58 °C); Biotinidase, 5′-ATC TAT GAA CAG CAA GTG ATG ACT-3′ (Tm = 66 °C) and 5′-AGG GAC CAG GGT GAA ATT GTC ATA-3′ (Tm = 70 °C); ;-actin: 5′-GGG TCA GAA TTC CTA TG-3′ and 5′-GGT CTC AAA CAT GAT CTG GG-3′ (Tm = 58 °C). PCR products were separated on 1% agarose gels and stained with ethidium bromide. The amount of PCR product was determined by densitometry by using a Fluor-S-imager (Bio-Rad) as previously described (3Pacheco-Alvarez D. Solórzano-Vargas R.S. Gravel R.A. Cervantes-Roldán R. Velázquez A. León-Del-Río A. J. Biol. Chem. 2004; 279: 52312-52318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 4Solórzano-Vargas R.S. Pacheco-Alvarez D. Leon-Del-Rio A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5325-5330Crossref PubMed Scopus (92) Google Scholar). The procedure was validated in prior studies by PCR amplification of different concentrations of cDNA fragments of HCS, biotinidase, and ;-actin (data not shown). The number of PCR cycles was also varied and plotted against fluorescence intensity to ensure that experiments were done within the exponential phase. For every experiment, the constitutive ;-actin mRNA was used as the reference cellular transcript. It was present at equivalent levels in all RNA samples. HCS Activity Assay Using p67 as Biotinylation Substrate—To determine HCS activity in normal and MCD-BD fibroblasts, we used a modification of the protocol described previously (16Leon-Del-Rio A. Gravel R.A. J. Biol. Chem. 1994; 269: 22964-22968Abstract Full Text PDF PubMed Google Scholar, 25Leon-Del-Rio A. Leclerc D. Akerman B. Wakamatsu N. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4626-4630Crossref PubMed Scopus (90) Google Scholar). Briefly, a pFLAG vector (Sigma) containing a cDNA fragment encoding the last 67 amino acids (640–703) of the ; subunit of human PCC was used to transform wild type E. coli XL1 Blue and E. coli C-124, a mutant strain unable to synthesize dethiobiotin, an essential intermediate in the production of biotin. Log phase XL-1 and C-124 cultures in L-broth medium were transferred to a biotin-free medium (7.5 mm (NH4)2SO4, 33 mm KH2PO4, 60 mm K2HPO4, 1.7 mm sodium citrate, 1 mm MgSO4, 0.2% dextrose, 0.1% casamino acids) and 2 mm isopropyl ;-d-thiogalactopyranoside and incubated at 37 °C for 4 h. The cells were sonicated three times with 10-s pulses and centrifuged at 15,000 rpm for 20 min. The proteins in the supernatant were resolved by 12% acrylamide gel electrophoresis (100 ;g of total protein/lane) and transferred to a PVDF membrane. Two biotinylated proteins are possible in cells expressing p67: p67, at 14 kDa, and BCCP, the 18-kDa subunit of E. coli ACC. To detect their positions on the gel, one lane containing proteins expressed by XL1 cells transformed with p67 was cut off from the membrane and incubated with streptavidin-AP to detect the biotinylated proteins (see Fig. 6A). The lower band, corresponding to p67, was used as a reference to cut out the section of the membrane in adjacent lanes containing unbiotinylated or apo-p67 expressed by E. coli C-124. The membrane pieces containing apo-p67 were used, in solid phase format, for HCS assays. HCS activity was monitored by incubating the membrane pieces for 1 h at 25 °C in 150 ;l of reaction buffer containing Tri-HCl, pH 8.0, 50 mm reduced glutathione, 22.5 mm MgCl2, 5 mm ATP, 1–3 ;Ci of [3H]biotin, and 100 ;g of total protein of crude extracts from normal or MCD-BD fibroblasts. For these experiments cells grown in biotin-supplemented medium were preincubated for 6 h with 1 ;m nonradioactive biotin and 63 ;m cycloheximide to block free biotinylation sites and prevent de novo carboxylases synthesis (5Pacheco-Alvarez D. Solórzano-Vargas R.S. González-Noriega A. Michalak C. Zempleni J. León-Del-Río A. Mol. Genet. Metab. 2005; 85: 301-307Crossref PubMed Scopus (31) Google Scholar). Crude extracts were prepared as previously described and passed twice through an Amicon ultra centrifugal device (Millipore) to eliminate nonradioactive biotin. The radioactive biotin incorporated into the membrane-bound p67 was estimated using a Beckman LS 6500 scintillation counter. Under these conditions, the p67 was in excess, and the assay was linear for the 1 h of incubation. cDNA and Genomic DNA Sequencing—To determine the mutations responsible for the phenotype of MCD-BD cells, the biotinidase cDNA was cloned in the pGEM vector (Promega). Biotinidase exons were amplified from genomic DNA as previously described (26Wolf B. Jensen K.P. Barshop B. Blitzer M. Carlson M. Goudie D.R. Gokcay G.H. Demirkol M. Baykal T. Demir F. Quary S. Shih L.Y. Pedro H.F. Chen T.H. Slonim A.E. Hum. Mutat. 2005; 25: 413Crossref PubMed Scopus (27) Google Scholar, 27Knight H. Reynolds T.R. Meyers G.A. Pomponio R.J. Buck G.A. Wolf B. Mamm. Genome. 1998; 9: 327-330Crossref PubMed Scopus (56) Google Scholar) and subcloned also in pGEM. Both cDNA and exons were sequenced at Laragen (Los Angeles, CA). Statistical Analysis—All of the experiments were done in triplicate and at least three different times. The results of biotin starvation on mRNA were normalized to ;-actin mRNA and expressed as a percentage of mRNA levels observed in cells grown in biotin-replete medium. The data are presented as the mean of three different experiments ± S.E. unless otherwise indicated. Statistical significance of p67 biotinylation results obtained with normal or MCD-BD cells were analyzed at 0.05 and 0.01 levels of significance using Student's t test one-way ANOVA. Molecular and Functional Characterization of the Biotinidase-deficient Cell Line MCD-BD—To characterize the cell line MCD-BD used as an experimental model in this study, we first identified the mutations responsible for biotinidase deficiency by sequencing the cDNA encoding this enzyme. This procedure resulted in the identification of a transversion 1330G → C, which causes a substitution of His for Asp444 (D444H), and a single base transition 511G → A, resulting in a substitution of Thr for Ala171 (A171T) (Fig. 2A). These mutations have been previously reported (26Wolf B. Jensen K.P. Barshop B. Blitzer M. Carlson M. Goudie D.R. Gokcay G.H. Demirkol M. Baykal T. Demir F. Quary S. Shih L.Y. Pedro H.F. Chen T.H. Slonim A.E. Hum. Mutat. 2005; 25: 413Crossref PubMed Scopus (27) Google Scholar) and are considered a common cause of profound biotinidase deficiency in children ascertained by newborn screening in the United States (28Norrgard K.J. Pomponio R.J. Swango K.L. Hymes J. Reynolds T. Buck G.A. Wolf B. Hum. Mutat. 1998; 11: 410Crossref PubMed Google Scholar). Because these mutations were originally described as a double mutation allele (28Norrgard K.J. Pomponio R.J. Swango K.L. Hymes J. Reynolds T. Buck G.A. Wolf B. Hum. Mutat. 1998; 11: 410Crossref PubMed Google Scholar), we sequenced all four biotinidase exons from genomic DNA to search for another mutation. This procedure confirmed the identified mutations and did not reveal the presence of any other mutation. To verify the impact of these mutations, we tested the biotinylation status of the carboxylases PC, PCC, and MCC in MCD-BD cells and compared the results with normal fibroblasts (positive control) and MCD-MK fibroblasts (negative control). The latter cells have unbiotinylated carboxylases in the standard biotin-replete medium caused by a homozygous, h" @default.
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• W2110134612 title "Impaired Biotinidase Activity Disrupts Holocarboxylase Synthetase Expression in Late Onset Multiple Carboxylase Deficiency" @default.
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