RESUMO
Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal ß-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid ß-oxidation via two enzymes with an overlapping substrate repertoire.
Assuntos
Ácidos Graxos , Oxirredução , Peroxissomos , Ácidos Graxos/metabolismo , Humanos , Peroxissomos/metabolismo , Mitocôndrias/metabolismo , Acil-CoA Desidrogenases/metabolismo , Acil-CoA Desidrogenases/genética , Animais , Células HEK293RESUMO
Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial ß-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5ß-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa , Modelos Moleculares , Especificidade por Substrato , Humanos , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/química , Cristalografia por Raios X , Domínio Catalítico , Acil-CoA Desidrogenases/metabolismo , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/química , Conformação Proteica , Sequência de AminoácidosRESUMO
Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.
Assuntos
Acil-CoA Desidrogenases , Erros Inatos do Metabolismo Lipídico , Humanos , Animais , Camundongos , Acil-CoA Desidrogenase/genética , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Acil-CoA Desidrogenases/genéticaRESUMO
Introduction: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Methods and results: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Discussion: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions.
Assuntos
Acil-CoA Desidrogenases , Síndromes de Imunodeficiência , Linfoma , Doenças da Imunodeficiência Primária , Humanos , Vesícula , Metabolismo Energético , Genótipo , Síndromes de Imunodeficiência/genética , Doenças da Imunodeficiência Primária/genética , Proteínas rab27 de Ligação ao GTP/genéticaRESUMO
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.
Assuntos
Acil-CoA Desidrogenases , Fibroblastos , Humanos , Camundongos , Animais , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , RNA Mensageiro/genética , Modelos Animais de Doenças , Fibroblastos/metabolismoRESUMO
Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), encodes a family of membrane proteins belonging to Resistance-Nodulation-Cell Division (RND) permeases also called multidrug resistance pumps. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in exporting of lipid components across the cell envelope. These proteins perform an essential role in MTB survival; however, there are no data regarding mutations in MmpL, polyketide synthase (PKS) and acyl-CoA dehydrogenase FadE proteins from Khyber Pakhtunkhwa, Pakistan. This study aimed to screen mutations in transmembrane transporter proteins including MmpL, PKS and Fad through whole-genome sequencing (WGS) in local isolates of Khyber Pakhtunkhwa province, Pakistan. Fourteen samples were collected from TB patients and drug susceptibility testing was performed. However, only three samples were completely sequenced. Moreover, 209 whole-genome sequences of the same geography were also retrieved from NCBI GenBank to analyze the diversity of mutations in MmpL, PKS and Fad proteins. Among the 212 WGS (Accession ID: PRJNA629298, PRJNA629388, and ERR2510337-ERR2510345, ERR2510546-ERR2510645), numerous mutations in Fad (n = 756), PKS (n = 479), and MmpL (n = 306) have been detected. Some novel mutations were also detected in MmpL, PKS and acyl-CoA dehydrogenase Fad. Novel mutations including Asn576Ser in MmpL8, Val943Gly in MmpL9 and Asn145Asp have been detected in MmpL3. The presence of a large number of mutations in the MTB membrane may have functional consequences on proteins. However, further experimental studies are needed to elucidate the variants' effect on MmpL, PKS and FadE functions.
Assuntos
Acil-CoA Desidrogenases , Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/farmacologia , Testes de Sensibilidade Microbiana , Mutação/genética , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/metabolismo , Acil-CoA Desidrogenases/farmacologiaRESUMO
Multiple-acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive disorder which can be split into three types. Type III MADD is associated with acute or subacute proximal muscle weakness and other variable non-specific features making it a challenging diagnosis for the clinician. This case report describes MADD in a 64 year-old lady, thought to be one of the latest first presentations of the disease. Unusually for this condition, the initial presentation was with dyspnoea. Furthermore, since this case provides further evidence that gene variants can predict age of onset, we advocate for further subclassification of type III MADD into late onset MADD (LO-MADD) when homozygous gene variants are present and very LO-MADD when heterozygous gene variants are found.
Assuntos
Acil-CoA Desidrogenases , Proteínas Ferro-Enxofre , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Feminino , Humanos , Pessoa de Meia-Idade , Flavoproteínas Transferidoras de Elétrons/genética , Flavoproteínas Transferidoras de Elétrons/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Proteínas Ferro-Enxofre/genética , Mutação , Deficiência Múltipla de Acil Coenzima A Desidrogenase/diagnóstico , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Acil-CoA Desidrogenases/genéticaRESUMO
Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.
Assuntos
Acil-CoA Desidrogenases , Fitosteróis , Pró-Fármacos , Fitosteróis/metabolismo , Oxirredutases/metabolismo , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Esteroides/metabolismo , Acil Coenzima A , Ácidos Carboxílicos , Cetosteroides , ÉsteresRESUMO
Cancer cells rewire the metabolic processes beneficial for cancer cell proliferation, survival, and their progression. In this study, metabolic processes related to glucose, glutamine, and fatty acid metabolism signatures were collected from the molecular signatures database and investigated in the context of energy metabolic pathways through available genome-wide expression profiles of liver cancer cohorts by gene sets-based pathway activation scoring analysis. The outcomes of this study portray that the fatty acid metabolism, transport, and its storage related signatures are highly expressed across early stages of liver tumors and on the contrary, the gene sets related to glucose transport and glucose metabolism are prominently activated in the hepatocellular carcinoma (HCC) stage. Based on the results, these metabolic pathways are clearly dysregulated across specific stages of carcinogenesis. The identified dimorphic metabolic pathway dysregulation patterns are further reconfirmed by examining corresponding metabolic pathway genes expression patterns across various stages encompassing profiles. Recurrence is the primary concern in the carcinogenesis of liver tumors due to liver tissues regeneration. Hence, to further explore these dysregulation effects on recurrent cirrhosis and recurrent HCC sample containing profile GSE20140 was examined and interestingly, this result also reiterated these differential metabolic pathways dysregulation. In addition, a recently established metabolome profile for the massive panel of cancer cell-lines, including liver cancer cell-lines, was used for further exploration. These findings also reassured those differential metabolites abundance of the fatty acid and glucose metabolic pathways enlighten those dimorphic metabolic pathways dysregulation. Moreover, ROC curves of fatty acid metabolic pathway genes such as acetyl-CoA carboxylase (ACACB), acyl-CoA dehydrogenase long chain (ACADL), and acyl-CoA dehydrogenase medium chain (ACADM) as well as glucose metabolic pathway genes such as phosphoglycerate kinase (PGK1), pyruvate dehydrogenase (PDHA1), pyruvate dehydrogenase kinase (PDK1) demonstrated greater sensitivity and specificity in the corresponding stage-specific tumors with significant p-values (p < 0.05). Furthermore, overall survival (OS) and recurrence-free survival (RFS) studies also reconfirmed that the rate-limiting genes expression of fatty acid and glucose metabolic pathways reveal better and poor survival in HCC patient cohorts, respectively. In conclusion, all these results clearly show that metabolic rewiring and the existence of two diverse metabolic pathways dysregulation involving fatty acid and glucose metabolism across the stages of liver tumors have been identified. These findings might be useful for developing therapeutic target treatments in stage-specific tumors.
Assuntos
Acil-CoA Desidrogenases , Carcinoma Hepatocelular , Neoplasias Hepáticas , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Acil-CoA Desidrogenases/metabolismo , Carcinogênese , Carcinoma Hepatocelular/patologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Redes e Vias Metabólicas , Oxirredutases , Fosfoglicerato Quinase/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , PiruvatosRESUMO
Altered thermal solubility measurement techniques are emerging as powerful tools to assess ligand binding, post-translational modification, protein-protein interactions, and many other cellular processes that affect protein state under various cellular conditions. Thermal solubility or stability profiling techniques are enabled on a global proteomic scale by employing isobaric tagging reagents that facilitate multiplexing capacity required to measure changes in the proteome across thermal gradients. Key among these is thermal proteomic profiling (TPP), which requires 8-10 isobaric tags per gradient and generation of multiple proteomic datasets to measure different replicates and conditions. Furthermore, using TPP to measure protein thermal stability state across different conditions may also require measurements of differential protein abundance. Here, we use the proteome integral stability alteration (PISA) assay, a higher throughput version of TPP, to measure global changes in protein thermal stability normalized to their protein abundance. We explore the use of this approach to determine changes in protein state between logarithmic and stationary phase Escherichia coli as well as glucose-starved human Hek293T cells. We observed protein intensity-corrected PISA changes in 290 and 350 proteins due to stationary phase transition in E. coli and glucose starvation, respectively. These data reveal several examples of proteins that were not previously associated with nutrient states by abundance alone. These include E. coli proteins such as putative acyl-CoA dehydrogenase (aidB) and chaperedoxin (cnoX) as well as human RAB vesicle trafficking proteins and many others which may indicate their involvement in metabolic diseases such as cancer.
Assuntos
Acil-CoA Desidrogenases , Proteínas de Escherichia coli , Humanos , Proteoma/metabolismo , Escherichia coli/metabolismo , Proteômica/métodos , Ligantes , Células HEK293 , Proteínas de Escherichia coli/metabolismo , Nutrientes , Glucose/metabolismo , Acil-CoA Desidrogenases/metabolismoRESUMO
Equine atypical myopathy (AM also referred to as multiple acyl-CoA dehydrogenases deficiency [MADD]) is thought to be caused by toxins metabolized from hypoglycin A (HGA) and méthylènecyclopropylglycine (MCPrG). HGA is contained in the seeds and seedlings of the sycamore tree (Acer pseudoplatanus); MCPrG has so far only been confirmed in seeds. Among other things, these substances can disrupt the fatty acids ß-oxidation pathway with the subsequent accumulation of certain acylcarnitines. The tentative diagnosis is based on anamnesis and clinical signs and can be verified by the detection of elevated creatine kinase activity, specific profile of acylcarnitines and the presence of HGA, MCPrG conjugates and/or their metabolites in peripheral blood and/or urine. Dry blood spots were collected for 15 days from a 3.5-year-old stallion which had been affected by AM and, as a control group, from twelve healthy horses. Two mass spectrometry methods were used for the analysis of 31 acylcarnitines, carnitine, HGA, MCPrG and their metabolites. HGA and six increased acylcarnitines were detected in the patient's blood throughout the monitoring period. Nine acylcarnitines were strongly correlated with HGA. Multivariate statistical analysis showed a clear separation of samples from the AM horse, where the metabolic profile tended to normalization in the later days after intoxication. Due to the longer persistence in the blood, the detection of HGA and elevated acylcarnitines profile appear to be an appropriate tool to confirm the diagnosis of AM, compared to metabolic products of HGA and MCPrG even in advanced cases.
Assuntos
Acil-CoA Desidrogenases , Doenças dos Cavalos , Doenças Musculares , Animais , Carnitina/análogos & derivados , Creatina Quinase , Ciclopropanos , Ácidos Graxos , Glicina/análogos & derivados , Doenças dos Cavalos/diagnóstico , Cavalos , Hipoglicinas , Masculino , Doenças Musculares/diagnóstico , Doenças Musculares/veterináriaRESUMO
Volume overload (VO) and pressure overload (PO) are two common pathophysiological conditions associated with cardiac disease. VO, in particular, often occurs in a number of diseases, and no clinically meaningful molecular marker has yet been established. We intend to find the main differential gene expression using bioinformatics analysis. GSE97363 and GSE52796 are the two gene expression array datasets related with VO and PO, respectively. The LIMMA algorithm was used to identify differentially expressed genes (DEGs) of VO and PO. The DEGs were divided into three groups and subjected to functional enrichment analysis, which comprised GO analysis, KEGG analysis, and the protein-protein interaction (PPI) network. To validate the sequencing data, cardiomyocytes from AR and TAC mouse models were used to extract RNA for qRT-PCR. The three genes with random absolute values of LogFC and indicators of heart failure (natriuretic peptide B, NPPB) were detected: carboxylesterase 1D (CES1D), whirlin (WHRN), and WNK lysine deficient protein kinase 2 (WNK2). The DEGs in VO and PO were determined to be 2761 and 1093, respectively, in this study. Following the intersection, 305 genes were obtained, 255 of which expressed the opposing regulation and 50 of which expressed the same regulation. According to the GO and pathway enrichment studies, DEGs with opposing regulation are mostly common in fatty acid degradation, propanoate metabolism, and other signaling pathways. Finally, we used Cytoscape's three techniques to identify six hub genes by intersecting 255 with the opposite expression and constructing a PPI network. Peroxisome proliferator-activated receptor (PPARα), acyl-CoA dehydrogenase medium chain (ACADM), patatin-like phospholipase domain containing 2 (PNPLA2), isocitrate dehydrogenase 3 (IDH3), heat shock protein family D member 1 (HSPD1), and dihydrolipoamide S-acetyltransferase (DLAT) were identified as six potential genes. Furthermore, we predict that the hub genes PPARα, ACADM, and PNPLA2 regulate VO myocardial changes via fatty acid metabolism and acyl-Coa dehydrogenase activity, and that these genes could be employed as basic biomarkers for VO diagnosis and treatment.
Assuntos
Acil-CoA Desidrogenases , Biologia Computacional , Animais , Biomarcadores , Biologia Computacional/métodos , Ácidos Graxos , Perfilação da Expressão Gênica/métodos , Camundongos , PPAR alfaRESUMO
The destiny of a messenger RNA is determined from a combination of in cis elements, like peculiar secondary structures, and in trans modulators, such as RNA binding proteins and non-coding, regulatory RNAs. RNA guanine quadruplexes belong to the first group: these strong secondary structures have been characterized in many mRNAs, and their stabilization or unwinding provides an additional step for the fine tuning of mRNA stability and translation. On the other hand, many cytoplasmic long non-coding RNAs intervene in post-transcriptional regulation, frequently by direct base-pairing with their mRNA targets. We have previously identified the lncRNA SMaRT as a key modulator of the correct timing of murine skeletal muscle differentiation; when expressed, lnc-SMaRT interacts with a G-quadruplex-containing region of Mlx-γ mRNA, therefore inhibiting its translation by counteracting the DHX36 helicase activity. The "smart" mode of action of lnc-SMaRT led us to speculate whether this molecular mechanism could be extended to other targets and conserved in other species. Here, we show that the molecular complex composed by lnc-SMaRT and DHX36 also includes other mRNAs. We prove that lnc-SMaRT is able to repress Spire1 translation through base-pairing with its G-quadruplex-forming sequence, and that Spire1 modulation participates to the regulation of proper skeletal muscle differentiation. Moreover, we demonstrate that the interaction between DHX36 and lnc-SMaRT is indirect and mediated by the mRNAs present in the complex. Finally, we suggest an extendibility of the molecular mechanism of lnc-SMaRT from the mouse model to humans, identifying potential functional analogues.
Assuntos
Diferenciação Celular/genética , Proteínas dos Microfilamentos/metabolismo , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Acil-CoA Desidrogenases , Animais , RNA Helicases DEAD-box , Quadruplex G , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/genética , RNA Mensageiro , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The human pathogen Mycobacterium tuberculosis depends on host fatty acids as a carbon source. However, fatty acid ß-oxidation is mediated by redundant enzymes, which hampers the development of antitubercular drugs targeting this pathway. Here, we show that rv0338c, which we refer to as etfD, encodes a membrane oxidoreductase essential for ß-oxidation in M. tuberculosis. An etfD deletion mutant is incapable of growing on fatty acids or cholesterol, with long-chain fatty acids being bactericidal, and fails to grow and survive in mice. Analysis of the mutant's metabolome reveals a block in ß-oxidation at the step catalyzed by acyl-CoA dehydrogenases (ACADs), which in other organisms are functionally dependent on an electron transfer flavoprotein (ETF) and its cognate oxidoreductase. We use immunoprecipitation to show that M. tuberculosis EtfD interacts with FixA (EtfB), a protein that is homologous to the human ETF subunit ß and is encoded in an operon with fixB, encoding a homologue of human ETF subunit α. We thus refer to FixA and FixB as EtfB and EtfA, respectively. Our results indicate that EtfBA and EtfD (which is not homologous to human EtfD) function as the ETF and oxidoreductase for ß-oxidation in M. tuberculosis and support this pathway as a potential target for tuberculosis drug development.
Assuntos
Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/metabolismo , Mycobacterium tuberculosis/metabolismo , Acil-CoA Desidrogenases/metabolismo , Animais , Modelos Animais de Doenças , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Óperon , Oxirredução , Oxirredutases/metabolismo , TuberculoseRESUMO
The biologically important, FAD-containing acyl-coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti-1,2-elimination of a proton and a hydride of aliphatic CoA thioesters. Here, we report on the structure and function of an ACAD from anaerobic bacteria catalyzing the unprecedented 1,4-elimination at C3 and C6 of cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA) to cyclohex-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) and at C3 and C4 of the latter to benzoyl-CoA. Based on high-resolution Ch1CoA dehydrogenase crystal structures, the unorthodox reactivity is explained by the presence of a catalytic aspartate base (D91) at C3, and by eliminating the catalytic glutamate base at C1. Moreover, C6 of Ch1CoA and C4 of Ch1,5CoA are positioned towards FAD-N5 to favor the biologically relevant C3,C6- over the C3,C4-dehydrogenation activity. The C1,C2-dehydrogenation activity was regained by structure-inspired amino acid exchanges. The results provide the structural rationale for the extended catalytic repertoire of ACADs and offer previously unknown biocatalytic options for the synthesis of cyclic 1,3-diene building blocks.
Assuntos
Acil-CoA Desidrogenases/metabolismo , Alcadienos/metabolismo , Acil-CoA Desidrogenases/química , Alcadienos/química , Biocatálise , Modelos Moleculares , Estrutura MolecularRESUMO
Mitochondrial complex I (CI) deficiencies (OMIM 252010) are the commonest inherited mitochondrial disorders in children. Acyl-CoA dehydrogenase 9 (ACAD9) is a flavoenzyme involved chiefly in CI assembly and possibly in fatty acid oxidation. Biallelic pathogenic variants result in CI dysfunction, with a phenotype ranging from early onset and sometimes fatal mitochondrial encephalopathy with lactic acidosis to late-onset exercise intolerance. Cardiomyopathy is often associated. We report a patient with childhood-onset optic and peripheral neuropathy without cardiac involvement, related to CI deficiency. Genetic analysis revealed compound heterozygous pathogenic variants in ACAD9, expanding the clinical spectrum associated to ACAD9 mutations. Importantly, riboflavin treatment (15 mg/kg/day) improved long-distance visual acuity and demonstrated significant rescue of CI activity in vitro.
Assuntos
Acil-CoA Desidrogenases/genética , Mutação da Fase de Leitura , Doenças do Nervo Óptico/tratamento farmacológico , Riboflavina/administração & dosagem , Idade de Início , Criança , Heterozigoto , Humanos , Masculino , Doenças do Nervo Óptico/genética , Riboflavina/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: We previously reported that ß-oxidation enzymes are present in the nucleus in close proximity to transcriptionally active promoters. Thus, we hypothesized that the fatty acid intermediate, butyryl-CoA, is the substrate for histone butyrylation and its abundance is regulated by acyl-CoA dehydrogenase short chain (ACADS). The objective of this study was to determine the genomic distribution of H3K9-butyryl (H3K9Bu) and its regulation by dietary fat, stress, and ACADS and its impact on gene expression. METHODS AND RESULTS: Using genome-wide chromatin immunoprecipitation-sequencing (ChIP-Seq), we show that H3K9Bu is abundant at all transcriptionally active promoters, where, paradoxically, it is most enriched in mice fed a fat-free vs high-fat diet. Deletion of fatty acid synthetase (FASN) abolished H3K9Bu in cells maintained in a glucose-rich but not fatty acid-rich medium, signifying that fatty acid synthesis from carbohydrates substitutes for dietary fat as a source of butyryl-CoA. A high-fat diet induced an increase in ACADS expression that accompanied the decrease in H3K9Bu. Conversely, the deletion of ACADS increased H3K9Bu in human cells and mouse hearts and reversed high-fat- and stress-induced reduction in promoter-H3K9Bu, whose abundance coincided with diminished stress-regulated gene expression as revealed by RNA sequencing. In contrast, H3K9-acetyl (H3K9Ac) abundance was minimally impacted by diet. CONCLUSION: Promoter H3K9 butyrylation is a major histone modification that is negatively regulated by high fat and stress in an ACADS-dependent fashion and moderates stress-regulated gene expression.
Assuntos
Acil-CoA Desidrogenases/metabolismo , Gorduras na Dieta/metabolismo , Histonas/metabolismo , Estresse Fisiológico , Acetilação , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The development of skeletal muscle is a highly ordered and complex biological process. Increasing evidence has shown that noncoding RNAs, especially long-noncoding RNAs (lncRNAs) and microRNAs, play a vital role in the development of myogenic processes. In this study, we observed that lncMyoD regulates myogenesis and changes myofiber-type composition. miR-370-3p, which is directly targeted by lncMyoD, promoted myoblast proliferation and inhibited myoblast differentiation in the C2C12 cell line, which serves as a valuable model for studying muscle development. In addition, the inhibition of miR-370-3p promoted fast-twitch fiber transition. Further analysis indicated that acyl-Coenzyme A dehydrogenase, short/branched chain (ACADSB) is a target gene of miR-370-3p, which is also involved in myoblast differentiation and fiber-type transition. Furthermore, our data suggested that miR-370-3p was sponged by lncMyoD. In contrast with miR-370-3p, lncMyoD promoted fast-twitch fiber transition. Taken together, our results suggest that miR-370-3p regulates myoblast differentiation and muscle fiber transition and is sponged by lncMyoD.
Assuntos
Acil-CoA Desidrogenases/genética , MicroRNAs/genética , Fibras Musculares de Contração Rápida/citologia , RNA Longo não Codificante/genética , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Camundongos , Desenvolvimento Muscular , Fibras Musculares de Contração Rápida/químicaRESUMO
The autotrophic 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle functions in thermoacidophilic, (micro)aerobic, hydrogen-oxidizing Crenarchaeota of the order Sulfolobales as well as in mesophilic, aerobic, ammonia-oxidizing Thaumarchaeota. Notably, the HP/HB cycle evolved independently in these two archaeal lineages, and crenarchaeal and thaumarchaeal versions differ regarding their enzyme properties and phylogeny. These differences result in altered energetic efficiencies between the variants. Compared to the crenarchaeal HP/HB cycle, the thaumarchaeal variant saves two ATP equivalents per turn, rendering it the most energy-efficient aerobic pathway for carbon fixation. Characteristically, the HP/HB cycle includes two enoyl coenzyme A (CoA) hydratase reactions: the 3-hydroxypropionyl-CoA dehydratase reaction and the crotonyl-CoA hydratase reaction. In this study, we show that both reactions are catalyzed in the aforementioned archaeal groups by a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase (Msed_2001 in crenarchaeon Metallosphaera sedula and Nmar_1308 in thaumarchaeon Nitrosopumilus maritimus). Although these two enzymes are homologous, they are closely related to bacterial enoyl-CoA hydratases and were retrieved independently from the same enzyme pool by the ancestors of Crenarchaeota and Thaumarchaeota, despite the existence of multiple alternatives. This striking similarity in the emergence of enzymes involved in inorganic carbon fixation from two independently evolved pathways highlights that convergent evolution of autotrophy could be much more widespread than anticipated.IMPORTANCE Inorganic carbon fixation is the most important biosynthetic process on Earth and the oldest type of metabolism. The autotrophic HP/HB cycle functions in Crenarchaeota of the order Sulfolobales and in ammonia-oxidizing Archaea of the phylum Thaumarchaeota that are highly abundant in marine, terrestrial, and geothermal environments. Bioinformatic prediction of the autotrophic potential of microorganisms or microbial communities requires identification of enzymes involved in autotrophy. However, many microorganisms possess several isoenzymes that may potentially catalyze the reactions of the cycle. Here, we studied the enzymes catalyzing 3-hydroxypropionyl-CoA dehydration and crotonyl-CoA hydration in Nitrosopumilus maritimus (Thaumarchaeota) as well as in Metallosphaera sedula (Crenarchaeota). We showed that both reactions were catalyzed by homologous promiscuous enzymes, which evolved independently from each other from their bacterial homologs. Furthermore, the HP/HB cycle is of applied value, and knowledge of its enzymes is necessary to transfer them to a heterologous host for synthesis of various value-added products.