dienoyl-CoA Reductase Research Articles

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5weeks of age and muscle atrophy by 11weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.

CRISPR/Cas9 is a powerful tool to edit the genome of the yeast Yarrowia lipolytica. Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using homologous directed recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an "excision approach" by simultaneously performing two DSBs in genes and reached 73 to 100% editing efficiency in double disruptions and 41.7% in a triple disruption. This work led to identifying SPS193 as a gene encoding a 2-4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.

Mitochondrial 2,4-dienoyl-CoA reductase (Decr) deficiency and impairment of thermogenesis in mouse brown adipose tissue

A large number of studies have demonstrated significance of polyunsaturated fatty acids (PUFAs) for human health. However, many aspects on signals translating PUFA-sensing into body homeostasis have remained enigmatic. To shed light on PUFA physiology, we have generated a mouse line defective in mitochondrial dienoyl-CoA reductase (Decr), which is a key enzyme required for β-oxidation of PUFAs. Previously, we have shown that these mice, whose oxidation of saturated fatty acid is intact but break-down of unsaturated fatty acids is blunted, develop severe hypoglycemia during metabolic stresses and fatal hypothermia upon acute cold challenge. In the current work, indirect calorimetry and thermography suggested that cold intolerance of Decr−/− mice is due to failure in maintaining appropriate heat production at least partly due to failure of brown adipose tissue (BAT) thermogenesis. Magnetic resonance imaging, electron microscopy, mass spectrometry and biochemical analysis showed attenuation in activation of lipolysis despite of functional NE-signaling and inappropriate expression of genes contributing to thermogenesis in iBAT when the Decr−/− mice were exposed to cold. We hypothesize that the failure in turning on BAT thermogenesis occurs due to accumulation of unsaturated long-chain fatty acids or their metabolites in Decr−/− mice BAT suppressing down-stream propagation of NE-signaling.

Evidence of Altered Mitochondrial Protein Expression After Chronic Ethanol Consumption in the Aged Estrogen-Deficient Female Rat Heart.

Estrogen loss has been implicated to increase the risk of alcoholic cardiomyopathy in postmenopausal women. The purpose of this study was to identify novel mitochondrial protein targets for the treatment of alcoholic cardiomyopathy in aged women using a state-of-the-art proteomic approach. We hypothesized that chronic ethanol (EtOH) ingestion exacerbates maladaptive mitochondrial protein expression in the aged female heart. Adult (3months) and aged (18months) F344 ovary-intact or ovariectomized (OVX) rats were randomly assigned an EtOH or control Lieber-DeCarli "all-liquid" diet for 20weeks. Proteomic analyses were conducted in mitochondria isolated from left ventricles using isobaric tags for relative and absolute quantification (iTRAQ) 8plex labeling and mass spectrometry (n=3 to 5/group). After EtOH, significant differences (false discovery rate <5%) were observed in electron transport chain components (NADH dehydrogenase [ubiquinone] flavoprotein 2) as well as proteins involved in lipid metabolism (2,4 dienoyl-CoA reductase) and cellular defense (catalase), suggesting a possible link to congestive heart failure. Directional changes in protein levels were confirmed by Western blotting. Additionally, EtOH significantly reduced state 3 mitochondrial respiration in all groups, yet only reduced respiratory control index in the aged OVX rat heart (p<0.05). Collectively, the data reveal that EtOH-induced changes in the mitochondrial proteome exacerbate cardiac dysfunction in aged and estrogen-deficient hearts, but not in adult. In conclusion, iTRAQ is a powerful tool for investigating new mitochondrial targets of alcoholic cardiomyopathy.

Investigation of the Effect of Herbicide Amiprophos Methyl on Spindle Formation and Proteome Change in Maize by Immunofluorescence and Proteomic Technique

To investigate the mechanism with which the phosphoric amide herbicide works, the effects of microtubule inhibiting herbicide amiprophos methyl (APM) on microtubule (MT) and proteomic were analyzed in the meristem cell and the mesocotyl, root, and leaves of maize. The immunofluorescence analysis showed that APM inhibited the spindle assembly in the cells during metaphase and anaphase when root meristems were treated with 10 μM APM for 12 h. Condensation of spindle, unequal spindle and multipolar spindle were observed, resulting in the formation of chromosome condensation in metaphase and unequal segregation of chormosome in anaphase. The frequency of these abnormal cells increased with the duration of treatment. Proteomics analysis revealed that 28 protein spots were altered, including the appearance of 15 new protein spots and the disappearance of 13 pre-existing proteins in the mesocotyl, root, and leaves. MALDI-TOF MS analysis of 10 protein spots has identified as cold acclimation protein WCOR615, ubiquitin, maturase K, an ubiquitin-like protein, Cytochrome P450 monooxygenase in mesocotyl, Ferrdoxins, 2,4 Dienoyl-CoA reductase in root, ATP-dependent protease, retrotransposn in leaves and an unnamed protein, which were involved in diverse biological processes, including protein synthesis and degradation, mRNA folding and splicing, cell cycle progression and signal transduction, energy metabolism and defense response. These results suggest that the herbicide APM is a specific chemical that interrupts microtubule dynamics and influences the expression of a number of proteins, which may be served as putative biochemical markers to monitor the toxicity of the herbicide in plants.

Newborn screening for disorders of fatty‐acid oxidation: experience and recommendations from an expert meeting

Experience with new-born screening (NBS) for disorders of fatty-acid oxidation (FAOD) is now becoming available from an increasing number of programs worldwide. The spectrum of FAOD differs widely between ethnic groups. Incidence calculations from reports from Australia, Germany, and the USA of a total of 5,256,999 newborns give a combined incidence of all FAOD of approximately 1:9,300. However, it appears to be much lower in Asians. Consequently, a significant prevalence and evidence for a clear benefit of NBS is proven for medium-chain acyl-CoA dehydrogenase deficiency (MCAD) only in countries with a high percentage of Caucasians, with very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) and long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency (LCHAD) being additional candidates. The long-term benefit for many disorders has still to be evaluated and will require international collaboration, especially for the rarest disorders. Short-chain acyl-CoA dehydrogenase deficiency (SCAD) [as well as Systemic carnitine transporter deficiency (CTD) and dienoyl-CoA reductase deficiency (DE-RED)] are conditions of uncertain clinical significance, but most FAOD have a spectrum of clinical presentations (healthy-death). Confirmatory diagnostic procedures should be agreed upon to ensure international comparability of results and evidence-based modifications. The case of short-chain acyl-CoA dehydrogenase deficiency (SCAD) deficiency shows that even inclusion of conditions without a clearly known natural course may prove useful with respect to gain of knowledge and consecutive exclusion of a biochemical abnormality without clinical significance, although this line of argument implies the existence of structured follow-up programs and bears ethical controversies. As a final conclusion, the accumulated evidence suggests all FAOD should to be included into tandem mass spectrometry (MS/MS)-based NBS programs provided sufficient laboratory performance is guaranteed.

Differential transcription of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo( a)pyrene

Benzo(a)pyrene (B( a)P) is a mutagenic polycyclic aromatic hydrocarbon (PAH) commonly released into the environment. B( a)P induces phase I biotransformation metabolism and peroxisome proliferation which is characterised in rodents by increased peroxisomal volume density, accompanied by the transcriptional induction of peroxisomal proteins. The aim of the present work was to study peroxisome proliferation at the transcriptional level, in comparison to the transcription of the well-characterised cytochrome P450 1A gene ( cyp1a) in the thicklip grey mullet Chelon labrosus. For this purpose, genes coding for the major peroxisomal membrane protein PMP70 and CYP1A were cloned using degenerate primers. Then juvenile mullets were single injected with B( a)P (5 mg/kg) and transcription of palmitoyl-CoA oxidase (aox1), multifunctional protein (mfp1), 3-ketoacyl-CoA thiolase (thio), Δ 2 ,Δ 4 dienoyl-CoA reductase 2, pmp70, catalase and cyp1a was semi-quantified in liver and gills 1 and 7 days after the injection. Transcription of aox1 and cyp1a was induced in gills 1 day after B( a)P injection. Cyp1a transcription was also induced in mullet liver one day after injection, indicating that B( a)P was available for the liver. This was further proved by the significant accumulation of B( a)P-like metabolites in bile 7 days after the injection. In liver, aox1, mfp1 and thio transcription was induced at day 1 followed by the induction of catalase transcription at day 7 that may reflect a response to an oxidative stress caused by B( a)P itself or by oxyradicals produced through the biotransformation metabolism and the peroxisomal β-oxidation. These hepatic responses were not reflected at AOX1 activity level. In conclusion, it has been shown for the first time that the three enzymes in the fish peroxisomal β-oxidation pathway are transcriptionally induced by B( a)P. It remains to be tested whether this enhanced transcription is reflected in an increase in the volume of peroxisomes.

Cloning and expression pattern of peroxisomal β-oxidation genes palmitoyl-CoA oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase in mussel Mytilus galloprovincialis and thicklip grey mullet Chelon labrosus

Due to the ability to respond after exposure to organic toxic compounds, peroxisome proliferation is used as biomarker of exposure to organic pollutants in mussels and in fish. Mussels are worldwide studied as sentinels of pollution in marine environments while mullets such as the thicklip grey mullet Chelon labrosus have been proposed as appropriate sentinel species since they inhabit highly polluted environments. In order to study genes of the inducible peroxisomal β-oxidation pathway in mussels Mytilus galloprovincialis and in C. labrosus, genes coding for the three enzymes in the inducible peroxisomal β-oxidation pathway, palmitoyl-CoA oxidase (AOX1), multifunctional protein (MFP1 in mullet and MFP2 in mussels), and 3-ketoacyl-CoA thiolase (THIO), were cloned. Additionally, a fragment of the peroxisomal Δ 2, Δ 4 dienoyl-CoA reductase 2 (DECR) necessary for the β-oxidation of unsaturated fatty acids was cloned in mullets. The whole open reading frame of aox1 sequenced in both mussels and mullets revealed high homology with known aox1 sequences, with highly conserved important domains such as the FAD binding motif or the typical peroxisomal targeting signal (PTS1). A thorough in silico analysis of the gene and genome databases allowed to identify in fish and molluscs sequence homologs of all the enzymes necessary for 2 of the 3 different paralog peroxisomal β-oxidation pathways described in metazoans (AOX1, AOX3, MFP1, MFP2, THIO and sterol carrier protein X). Only the enzyme necessary for the oxidation of branched chain fatty acids, AOX2, described in mammalian, avian and amphibian species, seems to be lacking from the genomes of fish and molluscs. In order to study the expression and regulation capacity of peroxisomal β-oxidation genes, aox1 and thio expression was determined in different tissues of mature and immature mullets and mussels collected in January and June, both genes being expressed higher in the digestive gland of mussels collected in June compared to January. Finally, in silico studies of the promoter regions in the piscine genomes available in the Ensembl genome repository, allowed the identification of putative peroxisome proliferator response elements that could explain the possible cellular and molecular mechanisms leading to peroxisome proliferation in fish. Further studies are needed to decipher molecular mechanisms of peroxisome proliferation in aquatic organisms under exposure to peroxisome proliferator xenobiotics.

Polymorphisms in positional candidate genes on BTA14 and BTA26 affect carcass quality in beef cattle

Several studies have reported the presence of carcass quality QTL on BTA14 and BTA26, with no specific genes being conclusively linked as their cause. The aim of this study was to identify polymorphisms in genes known to affect lipid metabolism in other species and to assess their association with carcass quality traits. Two genes located on BTA14, 2,4 dienoyl CoA reductase 1 (DECR1) and core binding factor, runt domain, alpha subunit 2, translocated to 1 gene (CBFA2T1), have been previously evaluated in other species and found to contain polymorphisms influencing lipid metabolism. A gene on BTA26, fibroblast growth factor 8 (FGF8), has in recent studies been linked to several QTL affecting obesity in mice, indicating its potential for regulating adiposity in other species. Sequencing analysis identified 9 polymorphisms in DECR1, 4 in CBFA2T1, and 4 in FGF8. Multiple sequence alignment of DECR1 among cattle, humans, and mice showed that 4 of these mutations lie in conserved regions across these species. Using 464 Angus, Charolais, and crossbred animals produced associations with ultrasound marbling score (CBFA2T1, P = 0.019), ultrasound backfat (DECR1, P = 0.012), carcass backfat (FGF8, P = 0.004), and lean meat yield (FGF8, P = 0.005). Quantitative trait loci analysis including a set of previously genotyped markers on BTA14, and 1 DECR1 polymorphism resulted in several significant QTL peaks: ultrasound backfat (UBF) at 91 cM, lean meat yield at 86 cM, carcass gradefat at 15 cM, and yield grade at 87 cM, all at the P < 0.05 level. Using DECR1 as a genetic covariate removed the UBF QTL, indicating that this SNP was contributing to the variation observed in UBF. A similar analysis was performed on BTA26 using 1 of the FGF8 polymorphisms. Results showed significant peaks for lean meat yield at 2 cM and for yield grade at 25 cM, both at P < 0.01, and for carcass backfat at 25 cM (P < 0.05). Removal of FGF8 SNP in further analysis resulted in the disappearance of the carcass backfat QTL. These results suggest that polymorphisms discovered in DECR1, CBFA2T1, and FGF8 may play a role in the lipid metabolism pathway affecting carcass quality traits in beef cattle. However, further studies are needed to confirm that these polymorphisms are responsible for the differences observed in carcass quality in beef cattle.

A note on associations between polymorphism within the 2,4-dienoyl-CoA reductase gene (&lt;i&gt;DECR1&lt;/i&gt;) and growth rate of Polish Landrace boars

The V54L missense mutation within the DECR1 gene, which encodes a mitochondrial 2,4- dienoylCoA reductase, was investigated to determine whether this polymorphism is associated with growth rate (daily gains), meat content and selection index in Polish Landrace boars kept under uniform feeding and environmental conditions (one herd). The genotype of 334 boars was determined by PCRRFLP, identifying 112, 162, and 60 boars bearing genotypes CC, CG and GG, respectively. Statistical analysis was carried out by the General Linear Model (GLM) procedure, including fi xed effects of DECR1 genotype, sire, and birth season. Signifi cant differences (P<0.01) between boars with CC and GG genotypes were found. Boars with genotype CC showed the highest daily gains (860.7 g ± 46.3), in comparison with boars bearing the GG genotype (841.7 g ± 53.6). The current fi ndings support the hypothesis that DECR1 V54L polymorphism is a promising marker of growth rate in pigs.

Mutational Analysis Reveals That All Tailoring Region Genes Are Required for Production of Polyketide Antibiotic Mupirocin by Pseudomonas fluorescens

The Pseudomonas fluorescens mupirocin biosynthetic cluster encodes six proteins involved in polyketide biosynthesis and 26 single polypeptides proposed to perform largely tailoring functions. In-frame deletions in the tailoring open reading frames demonstrated that all are required for mupirocin production. A bidirectional promoter region was identified between mupF, which runs counter to other open reading frames and its immediate neighbor macpC, implying the 74-kb cluster consists of two transcriptional units. mupD/E and mupJ/K must be cotranscribed as pairs for normal function implying co-assembly during translation. MupJ and K belong to a widely distributed enzyme pair implicated, with MupH, in methyl addition. Deletion of mupF, a putative ketoreductase, produced a mupirocin analogue with a C-7 ketone. Deletion of mupC, a putative dienoyl CoA reductase, generated an analogue whose structure indicated that MupC is also implicated in control of the oxidation state around the tetrahydropyran ring of monic acid. Double mutants with DeltamupC and DeltamupO, DeltamupU, DeltamupV, or DeltamacpE produced pseudomonic acid B but not pseudomonic acid A, as do the mupO, U, V, and macpE mutants, indicating that MupC must work after MupO, U, and V.

Restraint stress alters the duodenal expression of genes important for lipid metabolism in rat

Stress, such as trauma and injury, is known to cause transcriptional changes in various tissues; however, there is little information on tissue-specific gene expression in response to stress. Here, we have examined duodenal gene expression in rats subjected to whole-body immobilization in order to elucidate the mechanism underlying the stress response in the duodenum—one of the tissues that is most sensitive to external stress. DNA microarray analysis revealed that the immobilization for 2 weeks caused great changes in gene expression in the rat duodenum: 165 genes exhibited more than a two-fold change in expression level (103 up-regulated; 62 down-regulated). In addition, functional classification of these genes showed that immobilization preferentially stimulated the expression of genes related to lipid metabolism, including genes encoding mitochondrial HMG-CoA synthase, a key enzyme in ketogenesis; solute carrier 27A2, a fatty acid transporter; and dienoyl CoA reductase, a key enzyme in β-oxidation. To elucidate the factors mediating these immobilization-induced changes, we treated rats and small intestinal IEC-6 cells with dexamethasone and hydrogen peroxide. In both rats and IEC-6 cells, treatment with dexamethasone induced changes in gene expression that mimicked the immobilization-mediated increase in expression of the mitochondrial HMG-CoA synthase and dienoyl CoA reductase transcripts, suggesting that stress-induced synthesis of glucocorticoid hormones mediates, at least in part, the stress response in the duodenum. These results suggest that immobilization may alter lipid metabolism in the small intestine by modifying the expression of specific genes through which the small intestine may seek to protect itself from stress-induced damage.

The Mouse Gene PDCR Encodes a Peroxisomal Δ2,Δ4-Dienoyl-CoA Reductase

Here we describe the identification and characterization of a novel mouse gene, PDCR, that encodes a peroxisomal Delta(2), Delta(4)-dienoyl-CoA reductase. The mouse PDCR cDNA contains an 892-base pair open reading frame and is predicted to encode a 292-amino acid protein with a deduced molecular mass of 31,298 Da that terminates in a consensus type-1 peroxisomal targeting signal. Purified recombinant PDCR protein was generated from Escherichia coli and catalyzed the NADPH-dependent reduction of Delta(2)-trans, Delta(4)-trans-decadienoyl-CoA with a specific activity of 20 units/mg. Enzymatic characterization followed by high pressure liquid chromatography analysis of the products revealed that PDCR converted Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA to a Delta(3)-enoyl-CoA but not to a Delta(2)-enoyl-CoA. Kinetic analyses demonstrated that PDCR is active on a broad range of Delta(2), Delta(4)-dienoyl-CoAs. Although the observed substrate preference was to Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA, PDCR was also active on a C(22) substrate with multiple unsaturations, a result consistent with the role of peroxisomes in the oxidation of complex, very long chain, polyunsaturated fatty acids. The presence of a type-1 peroxisomal targeting signal Ala-Lys-Leu-COOH at the C terminus of PDCR suggested that this protein may be peroxisomal. We observed that tagged PDCR was efficiently transported to the peroxisome lumen in normal human fibroblasts but not in cells derived from a Zellweger syndrome patient with a specific defect in peroxisomal matrix protein import. We conclude that this protein resides within the peroxisome matrix and therefore represents the first mammalian peroxisomal Delta(2),Delta(4)-dienoyl-CoA reductase to be characterized at the molecular level.

Evidence for a peroxisomal fatty acid β‐oxidation involving d‐3‐hydroxyacyl‐CoAs

A novel D-(-)-3-hydroxyacyl-CoA hydro-lyase, forming 2-trans-enoyl-CoA and formerly designated as epimerase (EC 5.1.2.3), was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme, called D-3-hydroxyacyl-CoA hydro-lyase or D-specific 2-trans-enoyl-CoA hydratase, is shown to be required for the degradation of unsaturated fatty acids that contain double bonds extending from even-numbered C atoms. The D-3-hydroxyacyl-CoA hydro-lyase was exclusively localized within peroxisomes. A 10,000-fold purification by chromatography on a hydrophobic matrix, a cation exchanger, on hydroxyapatite and Mono S led to two proteins of apparent homogeneity, both exhibiting Mr of 65,000. The D-3-hydroxyacyl-CoA hydro-lyases are homodimers with slightly differing isoelectric points around pH = 9.0. They catalyze the conversion of 2-trans-enoyl-CoA into D-3-hydroxyacyl-CoA. The reverse reaction was observed but no reaction with 2-cis-enoyl-CoAs or L-3-hydroxyacyl-CoAs. 2-trans-Decenoyl-CoA was converted 10-times faster than 2-trans-butenoyl-CoA. The conversion of 4-cis-decenoyl-CoA into octenoyl-CoA was demonstrated in vitro with purified proteins with an assay mixture containing acyl-CoA oxidase, multifunctional protein, thiolase and the D-3-hydroxyacyl-CoA hydro-lyase. Comparisons of enzyme activities present in the cotyledons or isolated peroxisomes clearly show that the pathway via dienoyl-CoA reductase is much less effective than the sequence involving D-3-hydroxyacyl-CoA hydro-lyase.