Unique Behavior of Bacterially Expressed Rat Carnitine Palmitoyltransferase 2 and Its Catalytic Activity.

Mammalian type 2 carnitine parmitoyltransferase (EC 2.3.1.21), abbreviated as CPT2, is an enzyme involved in the translocation of fatty acid into the mitochondrial matrix space, and catalyzes the reaction acylcarnitine + CoA = acyl-CoA + carnitine. When rat CPT2 was expressed in Escherichia coli, its behavior was dependent on the presence or absence of i) its mitochondrial localization sequence and ii) a short amino acid sequence thought to anchor it to the mitochondrial inner membrane: CPT2 containing both sequences behaved as a hydrophobic protein, while recombinant CPT2 lacking both regions behaved as a water soluble protein; if only one region was present, the resultant proteins were observed in both fractions. Because relatively few protein species could be obtained from bacterial lysates as insoluble pellets under the experimental conditions used, selective enrichment of recombinant CPT2 protein containing both hydrophobic sequences was easily achieved. Furthermore, when CPT2 enriched in insoluble fraction was resuspended in an appropriate medium, it showed catalytic activity typical of CPT2: it was completely suppressed by the CPT2 inhibitor, ST1326, but not by the CPT1 inhibitor, malonyl-CoA. Therefore, we conclude that the bacterial expression system is an effective tool for characterization studies of mammalian CPT2.

CCN3 secretion is regulated by palmitoylation via ZDHHC22.

Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.

Lack of activation of the S113L variant of carnitine palmitoyltransfersase II by cardiolipin.

The phospholipid environment of the mitochondrial inner membrane, which contains large amounts of cardiolipin, could play a key role in transport of the long chain fatty acids. In the present study, the pre-incubation of cardiolipin with the wild type carnitine palmitoyltransferase (CPT) II led to a more than 1.5-fold increase of enzyme activity at physiological temperatures. At higher temperatures, however, there was a pronounced loss of activity. The most frequent variant S113L showed even at 37 °C a great activity loss. Pre-incubation of the wild type with both malonyl-CoA and cardiolipin counteracted the positive effect of cardiolipin. Malonyl-CoA, however, showed no inhibition effect on the variant in presence of cardiolipin. The activity loss in presence of cardiolipin at fever simulating situations was more pronounced for the variant comparing to the wild type. The reason might be a disturbed membrane association or a blockage of the active center of the mutated enzyme.

Malony-CoA inhibits the S113L variant of carnitine-palmitoyltransferase II.

Carnitine palmitoyltransferases (CPT), located both in the outer (CPT I) and inner membrane (CPT II) of mitochondria, are the key players for an efficient transport of long chain fatty acids into this cell compartment. The metabolite malonyl-CoA is known to inhibit CPT I, but not CPT II. His6-N-hCPT2 (wild type) and His6-N-hCPT2/ S113L (variant) were produced recombinantly in prokaryotic host, purified and characterized according to their functional and regulatory properties. The wild type and the variant showed the same enzymatic activity and were both inhibited by malonyl-CoA and malonate in a time-dependent manner. The inhibition was, however, significantly more pronounced in the mutated enzyme. The residual activities were 40% and 5% at temperatures of 4 °C and 30 °C, respectively. The inhibitory effect proceeded irreversibly with no recovery after postincubation of palmitoyl-CoA (Pal-CoA) as native substrate. A model of malonyl-CoA and malonate binding to human CPT II was suggested by docking studies to explain the action of the inhibitors regarding to the effect of the mutation on the protein conformation. Results indicated that not only CPT I, but also CPT II can be inhibited by malonyl-CoA. Thus, the complete inhibition of total CPT (i.e. CPT I and CPT II) in muscle homogenates by an established assay is not due to a lack of enzymatically active CPT II, but rather due to an abnormal regulation of the enzyme.

Differential in radiosensitizing potency of enantiomers of the fatty acid synthase inhibitor C75.

The elevated activity of fatty acid synthase has been reported in a number of cancer types. Inhibition of this enzyme has been demonstrated to induce cancer cell death and reduce tumor growth. In addition, the fatty acid synthase inhibitor drug C75 has been reported to synergistically enhance the cancer-killing ability of ionizing radiation. However, clinical use of C75 has been limited due to its producing weight loss, believed to be caused by alterations in the activity of carnitine palmitoyltransferase-1. C75 is administered in the form of a racemic mixture of (-) and (+) enantiomers that may differ in their regulation of fatty acid synthase and carnitine palmitoyltransferase-1. Therefore, we assessed the relative cancer-killing potency of different enantiomeric forms of C75 in prostate cancer cells. These results suggest that (-)-C75 is the more cytotoxic enantiomer and has greater radiosensitizing capacity than (+)-C75. These observations will stimulate the development of fatty acid synthase inhibitors that are selective for cancer cells and enhance the tumor-killing activity of ionizing radiation, while minimizing weight loss in cancer patients.

Carnosic acid attenuates obesity-induced glucose intolerance and hepatic fat accumulation by modulating genes of lipid metabolism in C57BL/6J-ob/ob mice.

BACKGROUND: Carnosic acid (CA), a major bioactive component of rosemary (Rosmarinus officinalis) leaves, is known to possess antioxidant and anti-adipogenic activities. In this study it was hypothesized that CA would ameliorate obesity-induced glucose intolerence and hepatic fat accumulation, and possible mechanisms are suggested. RESULTS: It was observed that a 0.02% (w/w) CA diet effectively decreased body weight, liver weight and blood triglyceride (TG) and total cholesterol levels (P < 0.05) compared with the control diet. CA at 0.02% significantly improved glucose tolerance, and hepatic TG accumulation was reduced in a dose-dependent manner. Hepatic lipogenic-related gene (L-FABP, SCD1 and FAS) expression decreased whereas lipolysis-related gene (CPT1) expression increased in animals fed the 0.02% CA diet (P < 0.05). Long-chain fatty acid content and the ratio of C18:1/C18:0 fatty acids were decreased in adipose tissue of animals fed the 0.02% CA diet (P < 0.05). Serum inflammatory mediators were also decreased significantly in animals fed the 0.02% CA diet compared with those of the obese control group (P < 0.05). CONCLUSION: These results suggest that CA is an effective anti-obesity agent that regulates fatty acid metabolism in C57BL/6J-ob/ob mice.

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism.

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid ß-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.

Structural characterization of the regulatory domain of brain carnitine palmitoyltransferase 1.

Neurons contain a mammalian-specific isoform of the enzyme carnitine palmitoyltransferase 1 (CPT1C) that couples malonyl-CoA to ceramide levels thereby contributing to systemic energy homeostasis and feeding behavior. In contrast to CPT1A, which controls the rate-limiting step of long-chain fatty acid ß-oxidation in all tissues, the biochemical context and regulatory mechanism of CPT1C are unknown. CPT1 enzymes are comprised of an N-terminal regulatory domain and a C-terminal catalytic domain (CD) that are separated by two transmembrane helices. In CPT1A, the regulatory domain, termed N, adopts an inhibitory and non-inhibitory state, Nα and Nß, respectively, which differ in their association with the CD. To provide insight into the regulatory mechanism of CPT1C, we have determined the structure of its regulatory domain (residues Met1-Phe50) by NMR spectroscopy. In relation to CPT1A, the inhibitory Nα state was found to be structurally homologues whereas the non-inhibitory Nß state was severely destabilized, suggesting a change in overall regulation. The destabilization of Nß may contribute to the low catalytic activity of CPT1C relative to CPT1A and makes its association with the CD unlikely. In analogy to the stabilization of Nß by the CPT1A CD, non-inhibitory interactions of N of CPT1C with another protein may exist.

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane.

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.

Molecular characterization, tissue distribution and kinetic analysis of carnitine palmitoyltransferase I in juvenile yellow catfish Pelteobagrus fulvidraco.

Up to date, only limited information is available on genetically and functionally different isoforms of CPT I enzyme in fish. In the study, molecular characterization and their tissue expression profile of three CPT Iα isoforms (CPT Iα1a, CPT Iα1b and CPT Iα2a) and a CPT Iß isoform from yellow catfish Pelteobagrus fulvidraco is determined. The activities and kinetic features of CPT I from several tissues have also been analyzed. The four CPT I isoforms in yellow catfish present distinct differences in amino acid sequences and structure. They are widely expressed in liver, heart, white muscle, spleen, intestine and mesenteric adipose tissue of yellow catfish at the mRNA level, but with the varying levels. CPT I activity and kinetics show tissue-specific differences stemming from co-expression of different isoforms, indicating more complex pathways of lipid utilization in fish than in mammals, allowing for precise control of lipid oxidation in individual tissue.

Screening of CPT1A-Targeting Lipid Metabolism Modulators Using Mitochondrial Membrane Chromatography.

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid ß-oxidation. Identifying the compounds targeting CPT1A warrants a promising candidate for modulating lipid metabolism. In this study, we developed a CPT1A-overexpressed mitochondrial membrane chromatography (MMC) to screen the compounds with affinity for CPT1A. Cells overexpressing CPT1A were cultured, and subsequently, their mitochondrial membrane was isolated and immobilized on amino-silica gel cross-linked by glutaraldehyde. After packing the mitochondrial membrane column, retention components of MMC were performed with LC/MS, whose analytic peaks provided structural information on compounds that might interact with mitochondrial membrane proteins. With the newly developed MMC-LC/MS approach, several Chinese traditional medicine extracts, such as Scutellariae Radix and Polygoni Cuspidati Rhizoma et Radix (PCRR), were analyzed. Five noteworthy compounds, baicalin, baicalein, wogonoside, wogonin, and resveratrol, were identified as enhancers of CPT1A enzyme activity, with resveratrol being a new agonist for CPT1A. The study suggests that MMC serves as a reliable screening system for efficiently identifying modulators targeting CPT1A from complex extracts.

An environment-dependent structural switch underlies the regulation of carnitine palmitoyltransferase 1A.

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid ß-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nß, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nß to be noninhibitory, with the relative Nα/Nß ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nß ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.

CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.

Packing of transmembrane domain 2 of carnitine palmitoyltransferase-1A affects oligomerization and malonyl-CoA sensitivity of the mitochondrial outer membrane protein.

The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 µM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.

Metabolomic profiling reveals a role for CPT1c in neuronal oxidative metabolism.

BACKGROUND: Carnitine Palmitoyltransferase-1c (CPT1c) is a neuron specific homologue of the carnitine acyltransferase family of enzymes. CPT1 isoenzymes transfer long chain acyl groups to carnitine. This constitutes a rate setting step for mitochondrial fatty acid beta-oxidation by facilitating the initial step in acyl transfer to the mitochondrial matrix. In general, neurons do not heavily utilize fatty acids for bioenergetic needs and definitive enzymatic activity has been unable to be demonstrated for CPT1c. Although there are studies suggesting an enzymatic role of CPT1c, its role in neurochemistry remains elusive. RESULTS: In order to better understand how CPT1c functions in neural metabolism, we performed unbiased metabolomic profiling on wild-type (WT) and CPT1c knockout (KO) mouse brains. Consistent with the notion that CPT1c is not involved in fatty acid beta-oxidation, there were no changes in metabolites associated with fatty acid oxidation. Endocannabinoids were suppressed in the CPT1c KO, which may explain the suppression of food intake seen in CPT1c KO mice. Although products of beta-oxidation were unchanged, small changes in carnitine and carnitine metabolites were observed. Finally, we observed changes in redox homeostasis including a greater than 2-fold increase in oxidized glutathione. This indicates that CPT1c may play a role in neural oxidative metabolism. CONCLUSIONS: Steady-state metabolomic analysis of CPT1c WT and KO mouse brains identified a small number of metabolites that differed between CPT1c WT and KO mice. The subtle changes in a broad range of metabolites in vivo indicate that CPT1c does not play a significant or required role in fatty acid oxidation; however, it could play an alternative role in neuronal oxidative metabolism.

Carnitine palmitoyl transferase I: Conformational changes induced by long-chain fatty acyl CoA ligands.

The Carnitine Palmitoyltranferase I (CPT1) catalyzes the rate-limiting step of long-chain fatty acid (LCFA) mitochondrial ß-oxidation. The enzyme promotes the conjugation of LCFA with l-carnitine, which allows LCFA to enter the mitochondria matrix. The structural features involved in CPT1 and LCFA-CoA interactions have not been fully elucidated, mainly due to the absence of CPT1 crystallographic data. Previous studies reported important residues (Lys556, Lys560, and Lys561) crucial to the CPT1 mechanism. Nonetheless, these studies have not explored the LCFA bindings. Using molecular modeling strategies, we aimed to understand the conformational changes in CPT1 structure induced by LCFA-CoA. For this purpose, a tridimensional CPT1A model was built by homology modeling using CRAT protein (PBD:1t7q, resolution 1.8 Å) as a template. We simulated the CPT1 structure in the presence and absence of LCFA-CoA by molecular dynamics (MD). By applying a principal component analysis (PCA), two states of apostructure CPT1 based on CoA-Loop (688-711) were observed. In contrast, just one state was evidenced along with smaller conformational subspaces in ligand-complexed simulations using LCFA-CoA. The CoA moiety of ligands interacts with charged residues, namely Lys560, Lys556, Arg563, and Arg645. The frequency of interactions observed for each of these residues is <60% of simulation time, suggesting a dynamic profile of interactions in synergy with long-chain carbon interactions over α-I (478-492). Collectively, these features may be associated with the catalytic conformation of LCFA-CoA to CPT1a. Further calculations of free-energy for different fatty acids, such as alpha-linolenic (ALA), gamma-linolenic (GLA), and arachidonic (ARA) acids, yielded energy values ranging from -76.9 ± 15.9 to -68.5 ± 10.0 kcal mol-1. In conclusion, the present structural model and simulations provide molecular-level insights into LCFA-CoA and CPT1a interactions. These findings may help to further knowledge on the conformational changes of CPT1a induced by LCFA-CoA derivates.

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling.

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8 microM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.

Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyl transferase I.

Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.

Genome duplication events have led to a diversification in the CPT I gene family in fish.

The enzyme carnitine palmitoyltransferase (CPT) I is a major regulator of mitochondrial fatty acid oxidation in vertebrates. Numerous genome duplication events throughout evolution have given rise to three (in mammals) or multiple (in fish) genetically and functionally different isoforms of this enzyme. In particular, these isoforms represent a diversification of kinetic and regulatory properties stemming from mutations at the genomic and proteomic levels. Phylogenetic reconstructions reveal a comprehensive view of the CPT I family in vertebrates and genomic modifications leading to structural changes in proteins and functional differences between tissues and taxa. In a model fish species (rainbow trout), the presence of five CPT I isoforms suggests repeated duplication events in bony fishes and salmonids. Subsequently, an array of nucleotide and amino acid substitutions in the isoforms may contribute to a tissue-specific and a previously observed species-specific difference in the IC(50) for malonyl-CoA. Moreover, all five isoforms are expressed in trout at the mRNA level in skeletal muscle, heart, liver, kidney, and intestine. In general, transcript levels of the beta-isoforms were higher in muscle tissues, while levels of the alpha-isoforms were higher in other tissues. Rainbow trout also exhibit developmental plasticity in relative mRNA expression of CPT I isoforms from fry to juvenile to adult stage. Thus the evolution of CPT I has resulted in a very diverse family of isoforms. These differences represent a degree of specificity in the ability of species to regulate function at the protein and tissue levels, which, in turn, may allow for precise control of lipid oxidation in individual tissues during physiological perturbations.

Structural insight into function and regulation of carnitine palmitoyltransferase.

The control of fatty acid translocation across the mitochondrial membrane is mediated by the carnitine palmitoyltransferase (CPT) system. Modulation of its functionality has simultaneous effects on fatty acid and glucose metabolism. This encourages use of the CPT system as drug target for reduction of gluconeogenesis and restoration of lipid homeostasis, which are beneficial in the treatment of type 2 diabetes mellitus and obesity. Recently, crystal structures of CPT-2 were determined in uninhibited forms and in complexes with inhibitory substrate-analogs with anti-diabetic properties in animal models and in clinical studies. The CPT-2 crystal structures have advanced understanding of CPT structure-function relationships and will facilitate discovery of novel inhibitors by structure-based drug design. However, a number of unresolved questions regarding the biochemistry and pharmacology of CPT enzymes remain and are addressed in this review.