Functional Studies of Yeast-Expressed Human Heart Muscle Carnitine Palmitoyltransferase I

Zhu, Hongfa; Shi, Jing; Vries, Yne de; Arvidson, Dennis N.; Cregg, James M.; Woldegiorgis, Gebre

doi:10.1006/abbi.1997.0314

Cited by 51 publications

(93 citation statements)

References 45 publications

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“…P. pastoris was chosen as an expression system for L-CPTI and the mutants, because it does not have endogenous CPT activity (6,(12)(13)(14)(15)(16). The P. pastoris expression plasmids expressed L-CPTI under control of the P. pastoris glyceraldehyde-3-phosphate dehydrogenase gene promoter (12,22).…”

Section: Resultsmentioning

confidence: 99%

“…As an enzyme that catalyzes the first rate-limiting step in ␤-oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA (1, 2), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis. Because of its central role in fatty acid metabolism, understanding the molecular mechanism of the regulation of the CPT system is an important first step in the development of treatments for diseases, such as myocardial ischemia and diabetes, and in human inherited CPTI deficiency diseases (9 -11).We developed a novel high level expression system for human heart M-CPTI, rat L-CPTI, and CPTII in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6,(12)(13)(14). Furthermore, by using this system, we have shown that CPTI and CPTII are active distinct enzymes and that L-CPTI and M-CPTI are distinct malonyl-CoA-sensitive CPTs that are reversibly inactivated by detergents.…”

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confidence: 99%

“…Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (3)(4)(5)(6)(7)(8). As an enzyme that catalyzes the first rate-limiting step in ␤-oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA (1, 2), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis.…”

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Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

Treber

Dai

Woldegiorgis

2003

Journal of Biological Chemistry

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Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine. To determine the role of the conserved glutamate residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.Carnitine palmitoyltransferase I (CPTI) 1 catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of L-carnitine, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, a rate-limiting step in ␤-oxidation (1, 2). Mammalian tissues express two isoforms of CPTI, a liver isoform (L-CPTI) and a muscle isoform (M-CPTI), that are 62% identical in amino acid sequence (3-8). As an enzyme that catalyzes the first rate-limiting step in ␤-oxidation, CPTI is regulated by its physiological inhibitor, malonyl-CoA (1, 2), the first intermediate in fatty acid synthesis, suggesting coordinated control of fatty acid oxidation and synthesis. Because of its central role in fatty acid metabolism, understanding the molecular mechanism of the regulation of the CPT system is an important first step in the development of treatments for diseases, such as myocardial ischemia and diabetes, and in human inherited CPTI deficiency diseases (9 -11).We developed a novel high level expression system for human heart M-CPTI, rat L-CPTI, and CPTII in the yeast Pichia pastoris, an organism devoid of endogenous CPT activity (6,(12)(13)(14). Furthermore, by using this system, we have shown that CPTI and CPTII are active distinct enzymes and that L-CPTI and M-CPTI are distinct malonyl-CoA-sensitive CPTs that are reversibly inactivated by detergents. Recent site-directed mutagenesis studies from our laboratory have demonstrated t...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

Treber

Dai

Woldegiorgis

2003

Journal of Biological Chemistry

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“…The purified antibodies recognized ACC2 in a Western blot and in an ELISA. The anti-human heart muscle M-CPT1 polyclonal antibodies were produced as described previously (25). mAbs (MAB1273) raised against the human mitochondrial 66-kDa protein were purchased from Chemicon.…”

Section: Methodsmentioning

confidence: 99%

The subcellular localization of acetyl-CoA carboxylase 2

Abu-Elheiga

Brinkley

Zhong

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

366

254

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Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), ACC1 (Mr ‫؍‬ 265 kDa) and ACC2 (Mr ‫؍‬ 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1-20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC-green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.A cetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an intermediate substrate that plays a pivotal role in the regulation of fatty acid metabolism. Besides its role in the biosynthesis of long-chain fatty acids (1-3), malonyl-CoA has been implicated in the regulation of the carnitine palmitoyl-CoA shuttle system that is involved in the mitochondrial ␤-oxidation of long-chain fatty acids. In animals, two isoforms of the carboxylase have been identified, ACC1 (M r ϭ 265,000) and ACC2 (M r ϭ 280,000) (4, 5). The two enzymes are encoded by separate genes and display distinct tissue distribution and regulation (6-9). The ACC1 carboxylases are highly expressed in lipogenic tissues, such as liver and adipose, and their levels are regulated transcriptionally while their activities are regulated posttranslationally by phosphorylation͞dephosphorylation of selected serine residues and by allosteric regulation through the action of citrate and palmitoyl-CoA (10-18). Dietary and hormonal states of the animal affect the level and activities of the ACC1 enzymes. A carbohydrate-rich, low-fat diet stimulates the expression and activities of ACC1, whereas starvation and diabetes reduce the ACC1 activities by repressing the expression of the ACC1 gene or by increasing the phosphorylation levels of the ACC1 protein (or both). Treating diabetic animals with insulin increases the activity of the enzyme either by dephosphorylation of the protein or by...

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“…This inverse relationship is retained in the recombinant proteins expressed in yeast (11,28,29) or mammalian cells (10). The present data suggest that it arises from the opposite effects of the amino-terminal segment, and the extreme amino terminus in particular, on these parameters in the two isoforms.…”

Section: Different Roles Of the Amino-terminal Segment In Determiningmentioning

confidence: 99%

Identification of Positive and Negative Determinants of Malonyl-CoA Sensitivity and Carnitine Affinity within the Amino Termini of Rat Liver- and Muscle-type Carnitine Palmitoyltransferase I

Jackson

Zammit

Price

2000

Journal of Biological Chemistry

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The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonylCoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 3 Ala) or ⌬(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant ⌬(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19 -82 of the enzyme. This was confirmed in the construct ⌬(19 -30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-⌬(19 -30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-⌬(19 -30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K 0.5 for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from ⌬(3-18) to ⌬(1-80). However, deletion of residues 3-18 from M-CPT I affected the K m for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme.Carnitine palmitoyltransferase I (CPT I, 1 malonyl-CoAsensitive) is an integral membrane protein first identified in the outer membrane and contact sites of mitochondria (1, 2). The enzyme catalyzes the formation of acylcarnitines from long-chain acyl-CoA esters, thus enabling the movement of acyl moieties across intracellular membranes. CPT I exists in two isoforms (Liver and Muscle), which have considerable sequence similarity but differ greatly, and inversely, in their malonyl-CoA sensitivity and K m for carnitine (3, 4). It is a polytopic protein, with two transmembrane (TM) segments and amino and carboxyl segments (approximately 46 and 652 residues, respectively) that are both exposed on the cytosolic aspect of the membrane (5). It has been shown that the extreme amino terminus of the nascent L-CPT I is retained in the mature protein (6) and, moreover, that it is essential for the expression of malonyl-CoA sensitivity (5, 7). Subsequent work with expressed CPT I constructs has confirmed these conclusions by showing that deletion of the amino-terminal highly conserved 6 amino acid residues of the L-isoform results in the loss of high affinity malonyl-CoA sensitivity (8). Glu-3, and to a much lesser extent His-5, have been identi...

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Functional Studies of Yeast-Expressed Human Heart Muscle Carnitine Palmitoyltransferase I

Cited by 51 publications

References 45 publications

Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

The subcellular localization of acetyl-CoA carboxylase 2

Identification of Positive and Negative Determinants of Malonyl-CoA Sensitivity and Carnitine Affinity within the Amino Termini of Rat Liver- and Muscle-type Carnitine Palmitoyltransferase I

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