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• W2041974026 abstract "Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid β-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 Å away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders. Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid β-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 Å away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders. Mitochondrial β-oxidation of long-chain fatty acids is a major source of energy production, especially during fasting, illness, or sustained exercise. Contrary to medium- and short-chain fatty acids that can cross the mitochondrial membranes by simple diffusion, long-chain fatty acids are imported into the mitochondrial matrix by the carnitine palmitoyltransferase (CPT, 1The abbreviations used are: CPTcarnitine palmitoyltransferaseCPT1Aliver isoform of human CPT1CATcarnitine acetyltransferaseTMtransmembranemtmutant.1The abbreviations used are: CPTcarnitine palmitoyltransferaseCPT1Aliver isoform of human CPT1CATcarnitine acetyltransferaseTMtransmembranemtmutant. EC 2.3.1.21) system (1.McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar, 2.Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (304) Google Scholar). The first component of this system is CPT1, an integral mitochondrial outer membrane protein, which catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine. CPT1 is tightly regulated by its physiological inhibitor malonyl-CoA, the first intermediate in fatty acid biosynthesis. This provides a mechanism for physiological regulation of β-oxidation in all mammalian tissues and for cellular fuel sensing based on the availability of fatty acids and glucose (1.McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar, 3.Prentki M. Corkey B.E. Diabetes. 1996; 45: 273-283Crossref PubMed Scopus (0) Google Scholar, 4.Zammit V.A. Biochem. J. 1999; 343: 505-515Crossref PubMed Scopus (99) Google Scholar). By its strategic metabolic position, CPT1 represents a potential drug target for the treatment of metabolic disorders such as diabetes, insulin resistance, and coronary heart disease (5.Ruderman N.B. Saha A.K. Vavvas D. Witters L.A. Am. J. Physiol. 1999; 276: E1-E18Crossref PubMed Google Scholar, 6.Unger R.H. Orci L. Faseb J. 2001; 15: 312-321Crossref PubMed Scopus (367) Google Scholar, 7.McGarry J.D. Diabetes. 2002; 51: 7-18Crossref PubMed Scopus (1210) Google Scholar). However, the rational design of pharmacological molecules for altering CPT1 activity requires a better understanding of its structure-function relationships. carnitine palmitoyltransferase liver isoform of human CPT1 carnitine acetyltransferase transmembrane mutant. carnitine palmitoyltransferase liver isoform of human CPT1 carnitine acetyltransferase transmembrane mutant. Three CPT1 isoforms with various tissue distribution and encoded by distinct genes have been identified (1.McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1327) Google Scholar, 2.Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (304) Google Scholar): a liver (CPT1A or L-CPT1) (8.Britton C.H. Schultz R.A. Zhang B. Esser V. Foster D.W. McGarry J.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1984-1988Crossref PubMed Scopus (125) Google Scholar), a muscle (CPT1B or M-CPT1) (9.Yamazaki N. Shinohara Y. Shima A. Yamanaka Y. Terada H. Biochim. Biophys. Acta. 1996; 1307: 157-161Crossref PubMed Scopus (102) Google Scholar), and a brain isoform (CPT1C) (10.Price N. van der Leij F. Jackson V. Corstorphine C. Thomson R. Sorensen A. Zammit V. Genomics. 2002; 80: 433-442Crossref PubMed Scopus (203) Google Scholar). During the past years, CPT1A has been the most investigated member of the acyltransferase family. CPT1A is anchored in the mitochondrial outer membrane by two transmembrane segments (TM1 and -2), its N terminus (residues 1–47) and C-terminal catalytic domain (residues 123–773) being located on the cytosolic face of mitochondria (11.Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1997; 323: 711-718Crossref PubMed Scopus (122) Google Scholar). The N-terminal domain (1–147 residues) was shown to be essential for mitochondrial import and for maintenance of a folded active and malonyl-CoA-sensitive conformation (12.Cohen I. Kohl C. McGarry J.D. Girard J. Prip-Buus C. J. Biol. Chem. 1998; 273: 29896-29904Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 13.Shi J. Zhu H. Arvidson D.N. Woldegiorgis G. J. Biol. Chem. 1999; 274: 9421-9426Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14.Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Functional analysis of natural and/or engineered mutations in CPT1A strongly contributed to understanding the catalytic and regulatory mechanisms implied in the acyltransferase family (15.Ijlst L. Mandel H. Oostheim W. Ruiter J.P. Gutman A. Wanders R.J. J. Clin. Invest. 1998; 102: 527-531Crossref PubMed Scopus (80) Google Scholar, 16.Morillas M. Gomez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17.Morillas M. Gomez-Puertas P. Rubi B. Clotet J. Arino J. Valencia A. Hegardt F.G. Serra D. Asins G. J. Biol. Chem. 2002; 277: 11473-11480Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 18.Morillas M. Gomez-Puertas P. Bentebibel A. Selles E. Casals N. Valencia A. Hegardt F.G. Asins G. Serra D. J. Biol. Chem. 2003; 278: 9058-9063Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19.Pan Y. Cohen I. Guillerault F. Feve B. Girard J. Prip-Buus C. J. Biol. Chem. 2002; 277: 47184-47189Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 20.Treber M. Dai J. Woldegiorgis G. J. Biol. Chem. 2003; 278: 11145-11149Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The recent three-dimensional structural models of the mouse and human carnitine acetyltransferase (CAT) provided critical insights into the molecular basis for fatty acyl chain transfer (21.Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 22.Wu D. Govindasamy L. Lian W. Gu Y. Kukar T. Agbandje-McKenna M. McKenna R. J. Biol. Chem. 2003; 278: 13159-13165Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). As CAT shares about 30–35% amino acid sequence identity to the other acyltransferases, its three-dimensional structure constitutes a more valuable tool than the model reported by Morillas et al. (16.Morillas M. Gomez-Puertas P. Roca R. Serra D. Asins G. Valencia A. Hegardt F.G. J. Biol. Chem. 2001; 276: 45001-45008Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) to understand the molecular mechanisms responsible for the deleterious effects of natural mutations in human CPT1A. CPT1A deficiency is a rare autosomal recessive disorder, characterized by severe episodes of hypoketotic hypoglycemia usually occurring after fasting or illness and beginning in early childhood (23.Bougneres P.F. Saudubray J.M. Marsac C. Bernard O. Odievre M. Girard J.R. N. Engl. J. Med. 1980; 302: 123-124Crossref PubMed Scopus (22) Google Scholar). To date, eleven missense mutations of the 17 mutations identified in more than 20 reported CPT1A-deficient patients were analyzed by exogenous expression (24.Bonnefont J.P. Demaugre F. Prip-Buus C. Saudubray J.M. Brivet M. Abadi N. Thuillier L. Mol. Genet. Metab. 1999; 68: 424-440Crossref PubMed Scopus (196) Google Scholar, 25.Brown N.F. Mullur R.S. Subramanian I. Esser V. Bennett M.J. Saudubray J.M. Feigenbaum A.S. Kobari J.A. Macleod P.M. McGarry J.D. Cohen J.C. J. Lipid Res. 2001; 42: 1134-1142Abstract Full Text Full Text PDF PubMed Google Scholar, 26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar, 27.Gobin S. Bonnefont J.P. Prip-Buus C. Mugnier C. Ferrec M. Demaugre F. Saudubray J.M. Rostane H. Djouadi F. Wilcox W. Cederbaum S. Haas R. Nyhan W.L. Green A. Gray G. Girard J. Thuillier L. Hum. Genet. 2002; 111: 179-189Crossref PubMed Scopus (41) Google Scholar, 28.Ogawa E. Kanazawa M. Yamamoto S. Ohtsuka S. Ogawa A. Ohtake A. Takayanagi M. Kohno Y. J. Hum. Genet. 2002; 47: 342-347Crossref PubMed Scopus (17) Google Scholar). Unfortunately, these mutations were not located within a structural model, impoverishing the informations they could bring regarding the structure-function relationships of this enzyme. In the present study, we investigated the molecular mechanisms responsible for the deleterious effects of five natural missense mutations identified in three CPT1A-deficient patients using both heterologous expression in Saccharomyces cerevisiae and a three-dimensional structure model of the human CPT1A that was created from our crystal structure of the mouse CAT (21.Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Molecular Analysis of CPT1A-deficient Patients—Informed consent was obtained from all subjects. Case report of patient 2 (29.Schaefer J. Jackson S. Taroni F. Swift P. Turnbull D.M. J. Neurol. Neurosurg. Psychiatry. 1997; 62: 169-176Crossref PubMed Scopus (42) Google Scholar) as well as molecular analysis of patients 1 and 3 (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar, 27.Gobin S. Bonnefont J.P. Prip-Buus C. Mugnier C. Ferrec M. Demaugre F. Saudubray J.M. Rostane H. Djouadi F. Wilcox W. Cederbaum S. Haas R. Nyhan W.L. Green A. Gray G. Girard J. Thuillier L. Hum. Genet. 2002; 111: 179-189Crossref PubMed Scopus (41) Google Scholar) have previously been reported. Fibroblasts from controls and patients were cultured as previously described (30.Saudubray J.M. Coude F.X. Demaugre F. Johnson C. Gibson K.M. Nyhan W.L. Pediatr. Res. 1982; 16: 877-881Crossref PubMed Scopus (75) Google Scholar) and were used to extract DNA according to standard methods and RNA using RNeasy Midi kit (Qiagen). Mutation analysis of patient 2 was performed at both cDNA and gDNA levels by sequencing approach, as previously described (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar, 27.Gobin S. Bonnefont J.P. Prip-Buus C. Mugnier C. Ferrec M. Demaugre F. Saudubray J.M. Rostane H. Djouadi F. Wilcox W. Cederbaum S. Haas R. Nyhan W.L. Green A. Gray G. Girard J. Thuillier L. Hum. Genet. 2002; 111: 179-189Crossref PubMed Scopus (41) Google Scholar). Construction of Human CPT1A Mutants—pYeCPT1A-A275T, -A414V, -Y498C, and -G709E were constructed with the QuikChange site-directed mutagenesis kit (Stratagene) using pYeCPT1A-WT (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar) as template that corresponds to the yeast expression vector pYeDP1/8–10 containing the full-length human CPT1A cDNA under control of the inducible GAL10 promoter. pYeCPT1A-A275T-A414V was constructed with a second step of mutagenesis creating the A414V mutation in pYeCPT1A-A275T. Mutations A275T, A414V, Y498C, and G709E were synthesized with pairs of mutagenized primers (sequences available upon request). cDNA of mutants were sequenced to assess the presence of the designed mutation as well as the absence of unwanted mutations. Plasmids were used to transform S. cerevisiae (haploid strain W303: MATα, his3, leu2, trp1, ura3, ade2–1, and can1–100) (31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Yeast Culture, Subcellular Fractionation, and Isolation of Yeast Mitochondria—Methods for yeast culture, subcellular fractionation, and isolation of yeast mitochondria were performed as previously described (31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Protein concentration was determined by the method of Lowry et al. (32.Lowry O.H. Rosebrough N.J. Lewis Farr A. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as standard. CPT Assay—CPT activity, apparent Km for carnitine and palmitoyl-CoA and IC50 value for malonyl-CoA, defined as the malonyl-CoA concentration that produces 50% inhibition of enzyme activity, were determined using mitochondria isolated from transformed yeasts, as previously reported (31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Assessment of the Folding State of Human CPT1A Mutants—Folding state of the human CPT1A mutants was analyzed by proteolytic digestion (10 μg/ml of trypsin) using intact or Triton X-100 (0.5% v/v) solubilized mitochondria (0.05 mg of protein/ml) as previously described for the rat protein (19.Pan Y. Cohen I. Guillerault F. Feve B. Girard J. Prip-Buus C. J. Biol. Chem. 2002; 277: 47184-47189Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar). Samples were analyzed by SDS-PAGE and immunoblotting. Western Blot Analysis—Proteins were analyzed by SDS-PAGE (33.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar) in an 8% gel and detected after blotting onto nitrocellulose as previously described (31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar) using the ECL detection system (Pierce) according to the supplier's instructions. Chemicals—TaqDNA polymerase, as well as PCR and sequencing reagents were purchased from Applied Biosystems. Yeast culture media products were from Difco, and Zymolase 20T was from ICN Biomedicals, Orsay, France. Others chemicals were purchased from Sigma. Molecular Analysis of Patient 2 and CPT1A Expression in Fibroblasts—As previously reported, three missense mutations were identified in patient 1: A275T and A414V carried on the paternal allele, and Y498C carried on the maternal allele (27.Gobin S. Bonnefont J.P. Prip-Buus C. Mugnier C. Ferrec M. Demaugre F. Saudubray J.M. Rostane H. Djouadi F. Wilcox W. Cederbaum S. Haas R. Nyhan W.L. Green A. Gray G. Girard J. Thuillier L. Hum. Genet. 2002; 111: 179-189Crossref PubMed Scopus (41) Google Scholar). The present molecular analysis of patient 2 permitted to identify both the heterozygous 2126G>A substitution predictive of the G709E mutation, and the 948delG deletion, which corresponds to the R316fsX328 frameshift at codon 316 (exon 9) generating a stop signal 12 codons downstream (exon 10) (Fig. 1A). This latter mutation was identified in a heterozygous state at the gDNA level, whereas it was not detected at the cDNA level (Fig. 1A), pointing out the instability of the R316fsX328 mRNA (Fig. 1B). CPT1A immunodetection in fibroblasts from patients indicated that, by contrast to a previously described patient homozygous for the G710E mutation (patient 3) (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar), neither patient 1 nor patient 2 expressed CPT1A protein at a detectable level (Fig. 2).Fig. 2Immunodetection of CPT1A in fibroblasts. Solubilized fibroblasts (100 μg of protein) from control (lane 1), patient 1 carrying the heterozygous A275T, A414V, and Y498C mutations (lane 2), patient 2 carrying the heterozygous G709E and R316fsX328 mutations (lane 3), and a previously described patient carrying the homozygous G710E mutation (lane 4) were analyzed by SDS-PAGE electrophoresis and immunoblotting using rat CPT1A and human β-actin antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Wild-type and CPT1A Mutants Expression in S. cerevisiae— The functional analysis of mutations A275T, A414V, Y498C, and G709E was performed using heterologous expression in yeast S. cerevisiae, an eukaryotic organism devoid of endogenous CPT1 activity (34.Brown N.F. Esser V. Foster D.W. McGarry J.D. J. Biol. Chem. 1994; 269: 26438-26442Abstract Full Text PDF PubMed Google Scholar), which was previously used for the functional analysis of the mutation G710E (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar). Subcellular fractionation experiments did not reveal any change in the mitochondrial targeting of the yeast-expressed wild-type and CPT1A mutants (results not shown). CPT1A immunodetection using variable amounts of mitochondria isolated from the different yeast strains show that proteins of predicted sizes were synthesized with different levels of expression (Fig. 3). Wild-type and mutants A275T and G709E were expressed at a similar steady-state level. By contrast, mutant Y498C exhibited a 2-fold lower protein level in comparison to wild-type, indicating that this mutation led to a slight protein instability. For mutants A414V and A275T-A414V, the level of protein expression was 20–30-fold lower than wild-type, indicating that the A414V substitution alone or in combination with A275T conferred a dramatic protein instability. Despite the fact that some of these mutations affect CPT1A protein stability, enough expressed proteins was recovered in yeast mitochondria, in contrast to what was observed in fibroblasts (Fig. 2), to perform the functional analysis of these substitutions. Enzyme Activity, Malonyl-CoA Inhibition, and Kinetic Properties of CPT1A Mutants—As shown in Table I, mutants A275T, Y498C, A414V, and A275T-A414V exhibited malonyl-CoA-sensitive CPT activity that was respectively 74, 46, 6, and 2% of that observed for the wild-type. For mutants Y498C, A414V, and A275T-A414V this decrease in CPT1 activity may partly result from the lower level of expressed protein (Fig. 3). As previously reported for mutant G710E (26.Prip-Buus C. Thuillier L. Abadi N. Prasad C. Dilling L. Klasing J. Demaugre F. Greenberg C.R. Haworth J.C. Droin V. Kadhom N. Gobin S. Kamoun P. Girard J. Bonnefont J.P. Mol. Genet. Metab. 2001; 73: 46-54Crossref PubMed Scopus (43) Google Scholar), mutant G709E was totally inactive whatever the concentration of substrate employed (Table I, Fig. 4, B and D) despite similar level of CPT1A protein expression when compared with wild-type (Fig. 3). Mutations A275T and Y498C did not alter malonyl-CoA sensitivity, their IC50 value for malonyl-CoA being similar to that of the wild-type (Table I). Due to the low residual activity in mutants A414V and A275T-A414V, it was not possible to assess their malonyl-CoA sensitivity. All mutants, except mutant G709E, exhibited normal saturation kinetics when the carnitine concentration varied relative to a fixed concentration of palmitoyl-CoA (Fig. 4, A and B) or when palmitoyl-CoA concentration varied when the molar ratio of palmitoyl-CoA/albumin was fixed at 6.1:1 (Fig. 4, C and D). Mutation A275T was previously characterized in COS cells as a functionally neutral polymorphism (25.Brown N.F. Mullur R.S. Subramanian I. Esser V. Bennett M.J. Saudubray J.M. Feigenbaum A.S. Kobari J.A. Macleod P.M. McGarry J.D. Cohen J.C. J. Lipid Res. 2001; 42: 1134-1142Abstract Full Text Full Text PDF PubMed Google Scholar). However, analysis of its saturation kinetics, which was not performed in the study of Brown et al. (25.Brown N.F. Mullur R.S. Subramanian I. Esser V. Bennett M.J. Saudubray J.M. Feigenbaum A.S. Kobari J.A. Macleod P.M. McGarry J.D. Cohen J.C. J. Lipid Res. 2001; 42: 1134-1142Abstract Full Text Full Text PDF PubMed Google Scholar), indicated that this mutation decreased by 25% to 43% the Vmax and catalytic efficiency (Vmax/Km) for carnitine and palmitoyl-CoA with no alteration in the apparent Km for both substrates (Table I). In comparison to wild-type, mutant Y498C had a similar apparent Km for palmitoyl-CoA but a 2-fold decrease in its apparent Km for carnitine, indicating a slight increased affinity of the enzyme to this substrate. Moreover, mutant Y498C showed a 3-fold decrease in its Vmax and catalytic efficiencies for carnitine and palmitoyl-CoA when compared with wild-type. Mutants A414V and A275T-A414V presented no alteration in the apparent Km for carnitine and palmitoyl-CoA, but at least a 98% decrease in their Vmax and catalytic efficiency whatever the substrate used (Table I). Thus, mutations A275T, Y498C, A414V, and A275T-A414V altered the Vmax and the catalytic efficiency more than the Km for carnitine and palmitoyl-CoA, whereas mutation G709E totally inactivated the enzyme.Table IEnzyme activity, malonyl-CoA inhibition and kinetic parameters of the wild-type and mutants CPT1A expressed in S. cerevisiaeStrainActivityMalonyl-CoACarnitinePalmitoyl-CoACatalytic efficiency-Malonyl-CoA+Malonyl-CoAIC50KmVmaxKmVmaxCarnitinePalmitoyl-CoAnmol/min/mgμmμmnmol/min/mgμmnmol/min/mgVmax/KmVmax/KmWild-type6.5 ± 0.60.8 ± 0.081.83 ± 0.4106.5 ± 8.4147.8 ± 10.582.8 ± 9.488.6 ± 10.21.4 (100%)1.07 (100%)A275T4.8 ± 0.30.4 ± 0.081.70 ± 0.32107.4 ± 16.887.0 ± 10.575.5 ± 8.361.1 ± 13.20.8 (60%)0.8 (76%)A414V0.4 ± 0.050.05 ± 0.03ND78.0 ± 8.01.6 ± 0.154.2 ± 4.21.1 ± 0.10.02 (1.5%)0.02 (1.9%)A275T-A414V0.13 ± 0.040.04 ± 0.02ND96.7 ± 2.90.6 ± 0.155.5 ± 3.50.4 ± 0.10.006 (0.5%)0.007 (0.7%)Y498C3.0 ± 0.20.2 ± 0.011.53 ± 0.0661.3 ± 6.829.2 ± 1.366.3 ± 9.525.9 ± 5.60.5 (34%)0.4 (37%)G709EUndetectableUndetectableNDNDNDNDNDNDND Open table in a new tab Assessment of the Folding State of CPT1A Mutants—Previous works (14.Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 19.Pan Y. Cohen I. Guillerault F. Feve B. Girard J. Prip-Buus C. J. Biol. Chem. 2002; 277: 47184-47189Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 31.Prip-Buus C. Cohen I. Kohl C. Esser V. McGarry J.D. Girard J. FEBS Lett. 1998; 429: 173-178Crossref PubMed Scopus (47) Google Scholar) showed that the rat CPT1A exhibits a native functional conformation characterized by a highly folded state resistant to trypsin proteolysis. When the outer mitochondrial membrane is disrupted, such as during the swelling procedure, trypsin is able to cleave the loop connecting TM1 and -2, hence generating an 82-kDa fragment. Moreover, the catalytic C-terminal domain of the rat CPT1A has been shown to contain a highly trypsin-resistant 60-kDa folded core that could be observed when solubilized mitochondria were submitted to trypsin proteolysis (14.Cohen I. Guillerault F. Girard J. Prip-Buus C. J. Biol. Chem. 2001; 276: 5403-5411Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). As shown in Fig. 5A, the human wild-type CPT1A protein also remained largely resistant to trypsin treatment in intact mitochondria. The integrity of the outer mitochondrial membrane was checked by the inaccessibility of cytochrome b2 to trypsin proteolysis (Fig. 5A). Mutants A275T and G710E, as well as mutants A414V and A275T-A414V (results not shown), exhibited the same protease resistance as the wild-type protein, whereas mutants Y498C and G709E were sensitive to trypsin proteolysis (Fig. 5A). When yeast mitochondria containing either the wild-type or the mutants A275T and G710E were solubilized by Triton X-100 in the presence of trypsin, both the 82- and 60-kDa fragments were detected (Fig. 5B, f1 and f2 fragments). These results strengthened the fact that the human CPT1A protein also contained within its catalytic C-terminal domain a highly folded trypsin-resistant core that was not affected by mutations A275T and G710E. By contrast, the generation of the f1 and f2 fragments was either less efficient or totally absent in the case of mutants Y498C and G709E (Fig. 5B), suggesting a partial unfolding of their C-terminal domain. The endogenous matrix soluble HSP70 protein (mtHSP70) was used as a positive control for trypsin proteolysis as its conformational states can be assessed by limited trypsin proteolysis (35.Fung K.L. Hilgenberg L. Wang N.M. Chirico W.J. J. Biol. Chem. 1996; 271: 21559-21565Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Localization of Mutations in a Structure Model of Human CPT1A—To understand the possible molecular mechanism for the effects of these mutations on the catalytic activity and the conformation of the enzyme, we examined their locations in a structure model of human CPT1A. The model was created with the program MODELLER (36.Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10448) Google Scholar) based on the crystal structure of the mouse CAT (21.Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), which shares 32% amino acid sequence identity with that of human CPT1A. Only residues 166–773 of human CPT1A have been used to built the structure model (Fig. 6A), as the first 160 residues of CPT1A do not have counterparts in CAT. This analysis indicates that residues Ala-275, Ala-414, Gly-709, and Gly-710 are in the core of the human CPT1A protein, whereas residue Tyr-498 is located in a surface loop which contains an inserted segment as compared with mouse CAT (Figs. 6A and 7).Fig. 7Sequence alignment of regions arrounding the human mutated CPT1A residues of various acyltransferases. Corresponding amino acids are boxed when conserved. Identical conserved residues are shaded. *, conserved His-277 in all malonyl-CoA sensitive enzymes; COT, carnitine octanoyltransferase; CAT, carnitine acetyltransferase; ChAT, chol" @default.
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• W2041974026 title "Functional and Structural Basis of Carnitine Palmitoyltransferase 1A Deficiency" @default.
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• W2041974026 cites W1858907820 @default.
• W2041974026 cites W1963817811 @default.
• W2041974026 cites W1973181474 @default.
• W2041974026 cites W1982013919 @default.
• W2041974026 cites W1982991739 @default.
• W2041974026 cites W1984197871 @default.
• W2041974026 cites W1984418909 @default.
• W2041974026 cites W1988632024 @default.
• W2041974026 cites W1988762815 @default.
• W2041974026 cites W1989280125 @default.
• W2041974026 cites W1997585363 @default.
• W2041974026 cites W2002827720 @default.
• W2041974026 cites W2004992318 @default.
• W2041974026 cites W2026577234 @default.
• W2041974026 cites W2031846075 @default.
• W2041974026 cites W2039531356 @default.
• W2041974026 cites W2047539110 @default.
• W2041974026 cites W2061539324 @default.
• W2041974026 cites W2062365107 @default.
• W2041974026 cites W2062644914 @default.
• W2041974026 cites W2064062425 @default.
• W2041974026 cites W2065283382 @default.
• W2041974026 cites W207208947 @default.
• W2041974026 cites W2074871036 @default.
• W2041974026 cites W2080001629 @default.
• W2041974026 cites W2084258426 @default.
• W2041974026 cites W2094328288 @default.
• W2041974026 cites W2098728010 @default.
• W2041974026 cites W2100837269 @default.
• W2041974026 cites W2105532009 @default.
• W2041974026 cites W2106457742 @default.
• W2041974026 cites W2127812547 @default.
• W2041974026 cites W2132730662 @default.
• W2041974026 cites W2430143578 @default.
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