acyl-CoA Esters Research Articles

Abstract B22: Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer

Abstract Lung cancer is the leading cause of cancer related deaths in the USA and worldwide. Lung tumorigenesis is a multistep process that involves several genetic aberrations. Activating mutations of the proto-oncogene KRAS (mutant KRAS) occur in ~30% of the cases of human non-small cell lung cancer (NSCLC), which is associated with aggressive, therapy-resistant disease. Despite the recent discovery of low affinity inhibitors, mutant KRAS is a challenging therapeutic target and there is a dearth of therapeutic options for these tumors. Mutant KRAS not only promotes tumorigenesis but also the survival of established lung cancer, both in mouse models and in certain human NSCLC lines. Therefore, in the absence of clinically-relevant effective inhibitors of mutant KRAS, there has been an intense clinical interest in the development of inhibitors of its downstream effectors. Importantly, mutant KRAS cancer cells undergo oncogene-directed metabolic reprogramming in order to meet the energetic and biosynthetic challenges of cell survival, growth and proliferation. Activation of certain pathways of fatty acid synthesis has been observed in many cancer types including lung cancer. Till date, fatty acid synthase (FASN) has been the candidate for drug development. Unfortunately, the inhibitors against FASN have poor pharmacokinetics and target related toxicity concerns. There is an urgent need for discovery of additional targets that inhibit lipid metabolism specifically in cancer cells that could be exploited for therapeutic gain. The goal of our study was to identify the cellular networks that mediate the maintenance of mutant KRAS lung cancer which further could be used as high priority therapeutic targets. To this end, we functionally analyzed the transcriptome of transgenic mouse lung tumors manipulated in vivo to undergo mutant Kras extinction, providing isogenic comparisons between mutant KRAS extinguished versus non-extinguished tumors for the discovery of new therapeutic targets. We determined that mutant KRAS controls tumor metabolism by regulating lipid homeostasis. We found that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrate for lipid synthesis and β-oxidation, is required for the survival of mutant KRAS lung cancer cells. These effects were not due to generalized toxicity, since we did not observe them in immortalized human bronchoalveolar cells and several NSCLC cells expressing wild type KRAS and in Acsl3 null mouse embryonic fibroblasts. We confirmed that ACSL3 is a mutant KRAS responsive gene expressed in the respiratory epithelium, in lung cancer cells and in primary human cancers. With mechanistic experiments we determined that mutant KRAS stimulates, in an ACSL3-dependent manner, the uptake and retention of fatty acids by lung cancer cells as well as their β-oxidation. As predicted by these experiments, ACSL3 is essential for the ability of mutant KRAS human lung cancer cells to form colonies in soft agar or to establish xenografts in immunocompromised mice. In addition, our preliminary results shows reduced tumor size and tumor burden in KrasG12D;Acsl3-/- mice. The detailed characterization of these mice is currently in progress. Our data demonstrate that mutant KRAS reprograms lipid homeostasis in lung cancer, establishing a cancer specific metabolic vulnerability. Thus, ACSL3 could be a viable therapeutic target for NSCLC driven by mutant KRAS. Citation Format: Mahesh S. Padanad, Georgia Konstantinidou, Chendong Yang, Margherita Melegari, Niranjan Venkateswaran, Kimberly Batten, Kenneth E. Huffman, Jerry W. Shay, John D. Minna, Ralph J. DeBerardinis, Pier P. Scaglioni. Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B22.

Carnitine palmitoyltransferase 1A functions to repress FoxO transcription factors to allow cell cycle progression in ovarian cancer

Cancer cells rely on hyperactive de novo lipid synthesis for maintaining malignancy. Recent studies suggest involvement in cancer of fatty acid oxidation, a process functionally opposite to lipogenesis. A mechanistic link from lipid catabolism to oncogenic processes is yet to be established. Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme of fatty acid β-oxidation (FAO) that catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine, thereby shuttling fatty acids into the mitochondrial matrix for β-oxidation. In the present study, we demonstrated that CPT1A was highly expressed in most ovarian cancer cell lines and primary ovarian serous carcinomas. Overexpression of CPT1A correlated with a poor overall survival of ovarian cancer patients. Inactivation of CPT1A decreased cellular ATP levels and induced cell cycle arrest at G0/G1, suggesting that ovarian cancer cells depend on or are addicted to CPT1A-mediated FAO for cell cycle progression. CPT1A deficiency also suppressed anchorage-independent growth and formation of xenografts from ovarian cancer cell lines. The cyclin-dependent kinase inhibitor p21WAF1 (p21) was identified as most consistently and robustly induced cell cycle regulator upon inactivation of CPT1A. Furthermore, p21 was transcriptionally upregulated by the FoxO transcription factors, which were in turn phosphorylated and activated by AMP-activated protein kinase and the mitogen-activated protein kinases JNK and p38. Our results established the oncogenic relevance of CPT1A and a mechanistic link from lipid catabolism to cell cycle regulation, suggesting that CPT1A could be a prognostic biomarker and rational target for therapeutic intervention of cancer.

Plant Cytosolic Acyl-CoA-Binding Proteins.

A gene family encoding six members of acyl-CoA-binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1-AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium- and long-chain acyl-CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane-associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser-scanning microscopy using autofluorescence-tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl-CoA esters suggested that they can potentially transfer oleoyl-CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non-plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters.

Deciphering the roles of acyl-CoA-binding proteins in plant cells.

Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed.

Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP−/− mice

Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication of the eye lid. We show that disruption of the Acbp gene leads to a significant enlargement of this gland with hypertrophy of the acinar cells and increased de novo synthesis of monoalkyl diacylglycerol, the main lipid species produced by the gland. Mice with conditional targeting of the Acbp gene in the epidermis recapitulate this phenotype, whereas generation of an artificial epidermal barrier during gland development reverses the phenotype. Our findings indicate that the Harderian gland is activated by the compromised epidermal barrier as an adaptive and protective mechanism to overcome the barrier defect.

Genome-wide identification and Phylogenic analysis of kelch motif containing ACBP in Brassica napus.

BackgroundAcyl-coA binding proteins (ACBPs) bind long chain acyl-CoA esters with very high affinity. Their possible involvement in fatty acid transportation from the plastid to the endoplasmic reticulum, prior to the formation of triacylglycerol has been suggested. Four classes of ACBPs were identified in Arabidopsis thaliana: the small ACBPs, the large ACBPs, the ankyrin repeats containing ACBPs and the kelch motif containing ACBPs. They differed in structure and in size, and showed multiple important functions. In the present study, Brassica napus ACBPs were identified and characterized.ResultsEight copies of kelch motif ACBPs were cloned, it showed that B. napus ACBPs shared high amino acid sequence identity with A. thaliana, Brassica rapa and Brassica oleracea. Furthermore, phylogeny based on domain structure and comparison map showed the relationship and the evolution of ACBPs within Brassicaceae family: ACBPs evolved into four separate classes with different structure. Chromosome locations comparison showed conserved syntenic blocks.ConclusionsACBPs were highly conserved in Brassicaceae. They evolved from a common ancestor, but domain duplication and rearrangement might separate them into four distinct classes, with different structure and functions. Otherwise, B. napus inherited kelch motif ACBPs from ancestor conserving chromosomal location, emphasizing preserved synteny block region. This study provided a first insight for exploring ACBPs in B. napus, which supplies a valuable tool for crop improvement in agriculture.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1735-6) contains supplementary material, which is available to authorized users.

Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus

Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.

Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

A novel role for central ACBP/DBI as a regulator of long-chain fatty acid metabolism in astrocytes.

Acyl-CoA-binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl-CoA esters. Several studies have suggested that ACBP acts as an acyl-CoA pool former and regulates long-chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam-Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism-related gene expression using ACBP-deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA-CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes. Acyl-CoA-binding protein (ACBP) or diazepam-binding inhibitor is a secreted peptide acting centrally as a GABAA allosteric modulator. Using brain slices, cortical, and hypothalamic astrocyte cultures from ACBP KO mice, we demonstrate that ACBP mainly localizes in astrocytes and regulates unsaturated but not saturated long-chain fatty acids (LCFA) metabolism. In addition, ACBP deficiency alters FA metabolism-related genes and results in intracellular FA accumulation while affecting their release. Our results support a novel role for ACBP in brain lipid metabolism. FA, fatty acids; KO, knockout; PL, phospholipids; TAG, triacylglycerol.

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development.

Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.

Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver

Acyl-CoA thioesterase (Acot)2 localizes to the mitochondrial matrix and hydrolyses long-chain fatty acyl-CoA into free FA and CoASH. Acot2 is expressed in highly oxi-dative tissues and is poised to modulate mitochondrial FA oxidation (FAO), yet its biological role is unknown. Using a model of adenoviral Acot2 overexpression in mouse liver (Ad-Acot2), we show that Acot2 increases the utilization of FA substrate during the daytime in ad libitum-fed mice, but the nighttime switch to carbohydrate oxidation is similar to control mice. In further support of elevated FAO in Acot2 liver, daytime serum ketones were higher in Ad-Acot2 mice, and overnight fasting led to minimal hepatic steatosis as compared with control mice. In liver mitochondria from Ad-Acot2 mice, phosphorylating O₂ consumption was higher with lipid substrate, but not with nonlipid substrate. This increase depended on whether FA could be activated on the outer mitochondrial membrane, suggesting that the FA released by Acot2 could be effluxed from mitochondria then taken back up again for oxidation. This circuit would prevent the build-up of inhibitory long-chain fatty acyl-CoA esters. Altogether, our findings indicate that Acot2 can enhance FAO, possibly by mitigating the accumulation of FAO intermediates within the mitochondrial matrix.

Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism.

Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis--phylogeny, site-directed mutagenesis, and expression in nonanther tissues.

Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C6) to very long-chain (C24) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C16-C18, and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation.

The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)-flame ionization detector (FID) and GC-mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs.

Subcellular localization of rice acyl-CoA-binding proteins (ACBPs) indicates that OsACBP6::GFP is targeted to the peroxisomes.

Acyl-CoA-binding proteins (ACBPs) show conservation at the acyl-CoA-binding (ACB) domain which facilitates binding to acyl-CoA esters. In Arabidopsis thaliana, six ACBPs participate in development and stress responses. Rice (Oryza sativa) also contains six genes encoding ACBPs. We investigated differences in subcellular localization between monocot rice and eudicot A. thaliana ACBPs. The subcellular localization of the six OsACBPs was achieved via transient expression of green fluorescence protein (GFP) fusions in tobacco (Nicotiana tabacum) epidermal cells, and stable transformation of A. thaliana. As plant ACBPs had not been reported in the peroxisomes, OsACBP6::GFP localization was confirmed by transient expression in rice sheath cells. The function of OsACBP6 was investigated by overexpressing 35S::OsACBP6 in the peroxisomal abc transporter1 (pxa1) mutant defective in peroxisomal fatty acid β-oxidation. As predicted, OsACBP1::GFP and OsACBP2::GFP were localized to the cytosol, and OsACBP4::GFP and OsACBP5::GFP to the endoplasmic reticulum (ER). However, OsACBP3::GFP displayed subcellular multi-localization while OsACBP6::GFP was localized to the peroxisomes. 35S::OsACBP6-OE/pxa1 lines showed recovery in indole-3-butyric acid (IBA) peroxisomal β-oxidation, wound-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) expression and jasmonic acid (JA) accumulation. These findings indicate a role for OsACBP6 in peroxisomal β-oxidation, and suggest that rice ACBPs are involved in lipid degradation in addition to lipid biosynthesis.

Measurement of lysophospholipid acyltransferase activities using substrate competition

Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoA-dependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. We describe here an alternative biochemical assay that provides a mixture of substrates presented to the microsomal extracts. Microsomal preparations from RAW 264.7 cells were used to compare traditional LPAT assays with data obtained using a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using LC-MS/MS. Both types of assays provided similar results, but the dual choice assay provided information about multiple fatty acyl chain incorporation into various phospholipid classes in a single reaction. Engineered suppression of LPCAT3 activity in RAW 264.7 cells was easily detected by the dual choice method. These findings demonstrate that this assay is both specific and sensitive and that it provides much richer biochemical detail than traditional assays.

A study on the chiral inversion of mandelic acid in humans.

Mandelic acid is a chiral metabolite of the industrial pollutant styrene and is used in chemical skin peels, as a urinary antiseptic and as a component of other medicines. In humans, S-mandelic acid undergoes rapid chiral inversion to R-mandelic acid by an undefined pathway but it has been proposed to proceed via the acyl-CoA esters, S- and R-2-hydroxy-2-phenylacetyl-CoA, in an analogous pathway to that for Ibuprofen. This study investigates chiral inversion of mandelic acid using purified human recombinant enzymes known to be involved in the Ibuprofen chiral inversion pathway. Both S- and R-2-hydroxy-2-phenylacetyl-CoA were hydrolysed to mandelic acid by human acyl-CoA thioesterase-1 and -2 (ACOT1 and ACOT2), consistent with a possible role in the chiral inversion pathway. However, human α-methylacyl-CoA racemase (AMACR; P504S) was not able to catalyse exchange of the α-proton of S- and R-2-hydroxy-2-phenylacetyl-CoA, a requirement for chiral inversion. Both S- and R-2-phenylpropanoyl-CoA were epimerised by AMACR, showing that it is the presence of the hydroxy group that prevents epimerisation of R- and S-2-hydroxy-2-phenylacetyl-CoAs. The results show that it is unlikely that 2-hydroxy-2-phenylacetyl-CoA is an intermediate in the chiral inversion of mandelic acid, and that the chiral inversion of mandelic acid is via a different pathway to that of Ibuprofen and related drugs.

Identification and biochemical characterization of five long-chain acyl-coenzyme A synthetases from the diatom Phaeodactylum tricornutum

Long-chain acyl-CoA synthetase (ACSL; EC 6.2.1.3) catalyzes the conversion of free fatty acid to acyl-CoA ester, which is necessary for many pathways of fatty acid and lipid metabolism. The diatom Phaeodactylum tricornutum genome encodes five putative ACSLs (PtACSL1-5) that contain several highly conserved motifs and share limited sequence similarities with each other and with other known ACSLs. To verify their long-chain acyl-CoA synthetase activities, five cDNAs encoding these PtACSLs were cloned, expressed, and tested for their ability to complement the Saccharomyces cerevisiae double mutant FAA1ΔFAA4Δ. Only two of five PtACSLs were able to restore growth, facilitate exogenous fatty acid uptake, and enhance storage lipid accumulation. We also found that P. tricornutum cells are capable of importing long-chain fatty acids from extracellular environment. The identification of P. tricornutum ACSLs will provide molecular basis for the study of ACSL-mediated lipid synthesis and metabolism in diatoms.

Aldehyde Decarbonylases: Enigmatic Enzymes of Hydrocarbon Biosynthesis

Aldehyde Decarbonylases: Enigmatic Enzymes of Hydrocarbon Biosynthesis

Aldehyde‐forming fatty acyl‐CoA reductase from cyanobacteria: expression, purification and characterization of the recombinant enzyme

Long-chain acyl-CoA reductases (ACRs) catalyze a key step in the biosynthesis of hydrocarbon waxes. As such they are attractive as components in engineered metabolic pathways for 'drop in' biofuels. Most ACR enzymes are integral membrane proteins, but a cytosolic ACR was recently discovered in cyanobacteria. The ACR from Synechococcus elongatus was overexpressed in Escherichia coli, purified and characterized. The enzyme was specific for NADPH and catalyzed the reduction of fatty acyl-CoA esters to the corresponding aldehydes, rather than alcohols. Stearoyl-CoA was the most effective substrate, being reduced more rapidly than either longer or shorter chain acyl-CoAs. ACR required divalent metal ions, e.g. Mg(2+), for activity and was stimulated ~ 10-fold by K(+). The enzyme was inactivated by iodoacetamide and was acylated on incubation with stearoyl-CoA, suggesting that reduction occurs through an enzyme-thioester intermediate. Consistent with this, steady state kinetic analysis indicates that the enzyme operates by a 'ping-pong' mechanism with kcat = 0.36 ± 0.023 min(-1), K(m)(stearoyl-CoA) = 31.9 ± 4.2 μM and K(m)(NADPH) = 35.6 ± 4.9 μM. The slow turnover number measured for ACR poses a challenge for its use in biofuel applications where highly efficient enzymes are needed.