scholarly journals Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal

2004 ◽  
Vol 379 (3) ◽  
pp. 777-784 ◽  
Author(s):  
Montserrat MORILLAS ◽  
Eduardo LÓPEZ-VIÑAS ◽  
Alfonso VALENCIA ◽  
Dolors SERRA ◽  
Paulino GÓMEZ-PUERTAS ◽  
...  
Keyword(s):  
Amino Acids ◽  
Structural Model ◽  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Carnitine Palmitoyltransferase ◽  
Carnitine Acetyltransferase ◽  
Catalytic Residues ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
D Model

CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of l-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered Km and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies.

scholarly journals Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-1) generated by alternative splicing of the CPT-Iβ gene

1998 ◽  
Vol 334 (1) ◽  
pp. 225-231 ◽  
Author(s):  
Geng-Sheng YU ◽  
Yi-Chun LU ◽  
Tod GULICK
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Topology ◽  
Splice Donor Site ◽  
The Novel ◽  
Pcr Screening ◽  
Mitochondrial Fatty Acid ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid β-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (α) or fat and muscle (β). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Iβ transcript, in addition to the β and α cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.

Cold acclimation increases carnitine palmitoyltransferase I activity in oxidative muscle of striped bass

1994 ◽  
Vol 266 (2) ◽  
pp. R405-R412 ◽  
Author(s):  
K. J. Rodnick ◽  
B. D. Sidell
Keyword(s):  
Cold Acclimation ◽  
Striped Bass ◽  
Fatty Acyl ◽  
Thermal Acclimation ◽  
Carnitine Palmitoyltransferase ◽  
Malonyl Coa ◽  
Red Muscle ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Long Chain

The effect of thermal acclimation on the activity of carnitine palmitoyltransferase I (CPT I), the rate-limiting enzyme for beta-oxidation of long-chain fatty acids, was determined in oxidative red muscle of striped bass (Morone saxatilis) acclimated at 5 or 25 degrees C. As observed in mammalian tissues, malonyl-CoA potently inhibited CPT I activity of mitochondria. Inhibition by malonyl-CoA required inclusions of both bovine serum albumin (BSA) and palmitoyl-CoA in the reaction media. Because BSA binds long-chain fatty acyl-CoAs, this observation suggests that free fatty acyl-CoAs may disrupt mitochondrial membranes and affect the CPT I protein. Cold acclimation increased citrate synthase activity 1.6-fold and total CPT activity 2-fold in homogenates of red muscle; free carnitine increased 62%, and specific activity of CPT I in mitochondria increased 2-fold. No differences were observed between cold- and warm-acclimated fish in substrate-binding properties of CPT I at an assay temperature of 15 degrees C, as judged by the Michaelis constant (Km) for carnitine (0.11 +/- 0.02 vs. 0.13 +/- 0.02 mM) or inhibition of CPT I, as determined by the half-maximal inhibition concentration (IC50) for malonyl-CoA (0.14 +/- 0.05 vs. 0.09 +/- 0.03 microM). Thermal sensitivity of CPT I (Q10 = 2.91 +/- 0.12 vs. 3.02 +/- 0.20) and preference of CPT I for different long-chain fatty acyl-CoA substrates (16:1-CoA = 16:0-CoA > 18:1-CoA) were not altered by thermal acclimation.(ABSTRACT TRUNCATED AT 250 WORDS)

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

2010 ◽  
Vol 298 (5) ◽  
pp. R1435-R1443 ◽  
Author(s):  
Xi Lin ◽  
Kwanseob Shim ◽  
Jack Odle
Keyword(s):  
Fatty Acid ◽  
Ketone Bodies ◽  
Carnitine Palmitoyltransferase ◽  
Acid Oxidation ◽  
Major Pathway ◽  
Malonyl Coa ◽  
Suckling Piglets ◽  
Carnitine Palmitoyltransferase I ◽  
Newborn Pigs ◽  
Cpt I

To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoyltransferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-14C]-palmitate oxidation was simultaneously measured. Accumulation rates of 14C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPT I for palmitoyl-CoA. Specifically, the IC50 for malonyl-CoA inhibition and Km value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8- and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced 14C accumulation in acetate ( P < 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-h-fed (72%) and 5-mo-old pigs (55%). The results demonstrate that acetate is the primary product of hepatic mitochondrial β-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I.

scholarly journals Evidence that the sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA is an important site of regulation of hepatic fatty acid oxidation in the fetal and newborn rabbit. Perinatal development and effects of pancreatic hormones in cultured rabbit hepatocytes

1990 ◽  
Vol 269 (2) ◽  
pp. 409-415 ◽  
Author(s):  
C Prip-Buus ◽  
J P Pegorier ◽  
P H Duee ◽  
C Kohl ◽  
J Girard
Keyword(s):  
Fatty Acid ◽  
Cyclic Amp ◽  
Fatty Acid Oxidation ◽  
Carnitine Palmitoyltransferase ◽  
Acid Oxidation ◽  
Fetal Hepatocytes ◽  
Malonyl Coa ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Hepatic Fatty Acid Oxidation

The temporal changes in oleate oxidation, lipogenesis, malonyl-CoA concentration and sensitivity of carnitine palmitoyltransferase I (CPT 1) to malonyl-CoA inhibition were studied in isolated rabbit hepatocytes and mitochondria as a function of time after birth of the animal or time in culture after exposure to glucagon, cyclic AMP or insulin. (1) Oleate oxidation was very low during the first 6 h after birth, whereas lipogenesis rate and malonyl-CoA concentration decreased rapidly during this period to reach levels as low as those found in 24-h-old newborns that show active oleate oxidation. (2) The changes in the activity of CPT I and the IC50 (concn. causing 50% inhibition) for malonyl-CoA paralleled those of oleate oxidation. (3) In cultured fetal hepatocytes, the addition of glucagon or cyclic AMP reproduced the changes that occur spontaneously after birth. A 12 h exposure to glucagon or cyclic AMP was sufficient to inhibit lipogenesis totally and to cause a decrease in malonyl-CoA concentration, but a 24 h exposure was required to induce oleate oxidation. (4) The induction of oleate oxidation by glucagon or cyclic AMP is triggered by the fall in the malonyl-CoA sensitivity of CPT I. (5) In cultured hepatocytes from 24 h-old newborns, the addition of insulin inhibits no more than 30% of the high oleate oxidation, whereas it stimulates lipogenesis and increases malonyl-CoA concentration by 4-fold more than in fetal cells (no oleate oxidation). This poor effect of insulin on oleate oxidation seems to be due to the inability of the hormone to increase the sensitivity of CPT I sufficiently. Altogether, these results suggest that the malonyl-CoA sensitivity of CPT I is the major site of regulation during the induction of fatty acid oxidation in the fetal rabbit liver.

Derivatives of R-Aminocarnitine without Ammonium Moiety as Liver Carnitine Palmitoyltransferase I (L-CPT I) Inhibitors

ChemMedChem ◽  
2011 ◽  
Vol 6 (11) ◽  
pp. 1977-1980 ◽  
Author(s):  
Emanuela Tassoni ◽  
Roberto Conti ◽  
Grazia Gallo ◽  
Silvia Vincenti ◽  
Lucilla Mastrofrancesco ◽  
...  
Keyword(s):  
Carnitine Palmitoyltransferase ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Derivatives Of

Myocardial carnitine palmitoyltransferase I expression and long-chain fatty acid oxidation in fetal and newborn lambs

2004 ◽  
Vol 286 (6) ◽  
pp. H2243-H2248 ◽  
Author(s):  
Beatrijs Bartelds ◽  
Janny Takens ◽  
Gioia B. Smid ◽  
Victor A. Zammit ◽  
Carina Prip-Buus ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Left Ventricular ◽  
Chain Fatty Acid ◽  
Carnitine Palmitoyltransferase ◽  
Long Chain Fatty Acid ◽  
Carnitine Palmitoyltransferase I ◽  
Cpt I ◽  
Long Chain

Carnitine palmitoyltransferase I (CPT I) catalyzes the conversion of acyl-CoA to acylcarnitine at the outer mitochondrial membrane and is a key enzyme in the control of long-chain fatty acid (LC-FA) oxidation. Because myocardial LC-FA oxidation increases dramatically after birth, we determined the extent to which CPT I expression contributes to these changes in the perinatal lamb. We measured the steady-state level of transcripts of the CPT1A and CPT1B genes, which encode the liver (L-CPT I) and muscle CPT I (M-CPT I) isoforms, respectively, as well as the amount of these proteins, their total activity, and the amount of carnitine in left ventricular tissue from fetal and newborn lambs. We compared these data with previously obtained myocardial FA oxidation rates in vivo in the same model. The results showed that CPT1B was already expressed before birth and that total CPT I expression transiently increased after birth. The protein level of M-CPT I was high throughout development, whereas that of L-CPT I was only transiently upregulated in the first week after birth. The total CPT I activity in vitro also increased after birth. However, the increase in myocardial FA oxidation measured in vivo (112-fold) by far exceeded the increase in gene expression (2.2-fold), protein amount (1.1-fold), and enzyme activity (1.2-fold) in vitro. In conclusion, these results stress the importance of substrate supply per se in the postnatal increase in myocardial FA oxidation. M-CPT I is expressed throughout perinatal development, making it a primary target for metabolic modulation of myocardial FA oxidation.

P5025Structural insights into catecholaminergic polymorphic ventricular tachycardia-associated RyR2 mutant channels using a three-dimensional in silico model

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Gao ◽  
T Makiyama ◽  
S Ohno ◽  
Y Yamamoto ◽  
Y Wuriyanghai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ventricular Tachycardia ◽  
In Silico ◽  
Structural Model ◽  
Three Dimensional ◽  
Polymorphic Ventricular Tachycardia ◽  
In Silico Modeling ◽  
Ryr2 Channel ◽  
The Relationship ◽  
Inherited Arrhythmias

Abstract Background The cardiac ryanodine receptors (RyR2) are large tetrameric calcium-permeant ion channels found in cardiac muscle sarcoplasmic reticulum, which play an important role in the control of intracellular Ca2+ release and cardiac contraction. Mutations in the RYR2 gene are associated with lethal arrhythmia diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) resulting from increased diastolic Ca2+ leak from mutant channels. RyR2 is a huge protein that each subunit of tetramer is comprised of 4967 amino acids, which hampers the detailed in vitro analysis of RyR2 mutant channels. Purpose We aimed to analyze the structural features of RyR2 mutant channels identified in our cohort with inherited arrhythmias using RyR2 three-dimensional (3D) in silico model to reveal the arrhythmogenic mechanisms. Methods A targeted next-generation sequencing panel for inherited arrhythmias was employed for genetic diagnosis of the patients. Then, we mapped the identified mutations on RyR2 3D structural model developed by cryo-EM images (PDB: 5go9, 5goa, Peng Science 2016) and investigated the relationship between the location of the mutations and specific functional sites. Results As a result of genetic analysis, we identified 93 RYR2 mutations from 112 probands with CPVT (n=93) or long-QT syndrome (LQTS) (n=19).64 of 93 (69%) RYR2 mutations are located in three “hot-spot” area (N-terminal (residues 77–466), central (2246–2534), and channel (3778–4959) hotspot. RyR2 3D in silico modeling revealed that the mutations are regionally distributed mainly in three parts: N-terminal, periphery, and channel part (Figure A). In N-terminal part (1–642 amino acid), 9 of 13 mutations alter the charges of the amino acids (Figure B). Especially, R169L, R169Q, and G172E are close to the interface between two neighboring subunits (∼20Å). These mutations which change the amino acid charge may cause a complete disruption of the ionic pair network and result in largest structural changes, which facilitates RyR2 channel opening. In periphery part (643–3528aa), 22 of 33 mutations are close to the two predicted binding sites of FKBP12.6, a stabilizer of RyR2 (∼5–40Å, Figure C). The mutations are supposed to disturb the binding affinity to the FKBP12.6 resulting in RyR2 channel instability. In channel part (3613–4968aa), 16 of 40 mutations are located near two interface. (FigureD) 12 mutations are close to the Ca2+ sensor and the other 4 mutations are adjacent to the pore-forming segment. Especially, V4821I is just located on this segment and strongly expected to impair the channel function. Above all, RyR2 3D in silico modeling revealed that 63 of all 93 (68%) identified mutations are supposed to be pathogenic. Location of RYR2 mutations in 3D model Conclusion 3D structural model of RyR2 is useful for the investigation of the pathogenic mechanisms of CPVT-related mutations. Further studies are needed to elucidate the relationship between the location of the mutations and clinical phenotypes.

scholarly journals Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene

1998 ◽  
Vol 330 (1) ◽  
pp. 217-224 ◽  
Author(s):  
A. Edwards PARK ◽  
L. Michelle STEFFEN ◽  
Shulan SONG ◽  
M. Vicki PARK ◽  
A. George COOK
Keyword(s):  
Genomic Library ◽  
Transcriptional Start Site ◽  
Carnitine Palmitoyltransferase ◽  
Proximal Promoter ◽  
Exon 2 ◽  
Exon 1 ◽  
Carnitine Palmitoyltransferase I ◽  
Specific Regulation ◽  
Cpt I ◽  
I Gene

Carnitine palmitoyltransferase I (CPT I) catalyses the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. The cDNAs of two isoforms of CPT I, termed the hepatic and muscle isoforms, have been cloned. Expression of the hepatic CPT I gene (L-CPT I) is subject to developmental, hormonal and tissue specific regulation. We have cloned the promoter of the L-CPT I gene from a rat genomic library. In the L-CPT I gene, there are two exons 5ʹ to the exon containing the ATG that initiates translation. Exon 1 and the 5ʹ end of exon 2 contain sequences that were not previously described in the rat L-CPT I cDNA. There is an alternatively spliced form of the L-CPT I mRNA in which exon 2 is skipped. The proximal promoter of the L-CPT I gene is extremely GC rich and does not contain a TATA box. There are several putative Sp1 binding sites near the transcriptional start site. A 190 base pair fragment of the promoter can efficiently drive transcription of luciferase and CAT (chloramphenicol acetyltransferase) reporter genes transiently transfected into HepG2 cells. Sequences in both the first intron and the promoter contribute to basal expression. Our results provide the foundation for further studies into the regulation of L-CPTI gene expression.

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