RESUMEN
Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprograming, in which EIN2-C-directed regulation of histone acetylation is the first key step for chromatin to perceive ethylene signaling. But how the nuclear acetyl coenzyme A (acetyl CoA) is produced to ensure the ethylene-mediated histone acetylation is unknown. Here we report that ethylene triggers the accumulation of the pyruvate dehydrogenase complex (PDC) in the nucleus to synthesize nuclear acetyl CoA to regulate ethylene response. PDC is identified as an EIN2-C nuclear partner, and ethylene triggers its nuclear accumulation. Mutations in PDC lead to an ethylene hyposensitivity that results from the reduction of histone acetylation and transcription activation. Enzymatically active nuclear PDC synthesizes nuclear acetyl CoA for EIN2-C-directed histone acetylation and transcription regulation. These findings uncover a mechanism by which PDC-EIN2 converges the mitochondrial enzyme-mediated nuclear acetyl CoA synthesis with epigenetic and transcriptional regulation for plant hormone response.
Asunto(s)
Acetilcoenzima A , Proteínas de Arabidopsis , Arabidopsis , Núcleo Celular , Etilenos , Regulación de la Expresión Génica de las Plantas , Histonas , Complejo Piruvato Deshidrogenasa , Acetilación , Etilenos/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Acetilcoenzima A/metabolismo , Transcripción Genética , Mutación , Transducción de Señal , Receptores de Superficie CelularRESUMEN
Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.
Asunto(s)
Represión Catabólica , Pseudomonas putida , Complejo Piruvato Deshidrogenasa , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Pseudomonas putida/enzimología , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Hidrocarburos Aromáticos/metabolismo , Biodegradación Ambiental , Acetilcoenzima A/metabolismo , Ácido Pirúvico/metabolismo , Eliminación de Gen , Redes y Vías Metabólicas/genéticaRESUMEN
Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation that links glycolysis-derived pyruvate with the tricarboxylic acid (TCA) cycle. Although skeletal muscle is a significant site for glucose oxidation and is closely linked with metabolic flexibility, the importance of muscle PDH during rest and exercise has yet to be fully elucidated. Here, we demonstrate that mice with muscle-specific deletion of PDH exhibit rapid weight loss and suffer from severe lactic acidosis, ultimately leading to early mortality under low-fat diet provision. Furthermore, loss of muscle PDH induces adaptive anaplerotic compensation by increasing pyruvate-alanine cycling and glutaminolysis. Interestingly, high-fat diet supplementation effectively abolishes early mortality and rescues the overt metabolic phenotype induced by muscle PDH deficiency. Despite increased reliance on fatty acid oxidation during high-fat diet provision, loss of muscle PDH worsens exercise performance and induces lactic acidosis. These observations illustrate the importance of muscle PDH in maintaining metabolic flexibility and preventing the development of metabolic disorders.
Asunto(s)
Acidosis Láctica , Alanina , Músculo Esquelético , Complejo Piruvato Deshidrogenasa , Ácido Pirúvico , Animales , Ratones , Acidosis Láctica/fisiopatología , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Glutamina/metabolismo , Alanina/metabolismo , Eliminación de Gen , Dieta , Mortalidad PrematuraRESUMEN
Candida albicans, the most prevalent fungal pathogen in the human microbiota can form biofilms on implanted medical devices. These biofilms are tolerant to conventional antifungal drugs and the host immune system as compared to the free-floating planktonic cells. Several in vitro models of biofilm formation have been used to determine the C. albicans biofilm-forming process, regulatory networks, and their properties. Here, we performed a genome-wide transcript profiling with C. albicans cells grown in YPD medium both in planktonic and biofilm condition. Transcript profiling of YPD-grown biofilms was further compared with published Spider medium-grown biofilm transcriptome data. This comparative analysis highlighted the differentially expressed genes and the pathways altered during biofilm formation. In addition, we demonstrated that overexpression of the PDB1 gene encoding a subunit of the pyruvate dehydrogenase resulted in defective biofilm formation. Altogether, this comparative analysis of transcript profiles from two different studies provides a robust reading on biofilm-altered genes and pathways during C. albicans biofilm development.
Asunto(s)
Candida albicans , Complejo Piruvato Deshidrogenasa , Humanos , Candida albicans/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Antifúngicos/metabolismo , Transcriptoma , BiopelículasRESUMEN
Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia resulting in high morbidity and mortality, with limited therapeutic options. The E1 component of the mitochondrial multienzyme PDC (PDC-E1) is a symmetric dimer of heterodimers (αß/α'ß') encoded by the PDHA1 and PDHB genes, with two symmetric active sites each consisting of highly conserved phosphorylation loops A and B. PDHA1 mutations are responsible for 82-88% of cases. Greater than 85% of E1α residues with disease-causing missense mutations (DMMs) are solvent-inaccessible, with â¼30% among those involved in subunit-subunit interface contact (SSIC). We performed molecular dynamics simulations of wild-type (WT) PDC-E1 and E1 variants with E1α DMMs at R349 and W185 (residues involved in SSIC), to investigate their impact on human PDC-E1 structure. We evaluated the change in E1 structure and dynamics and examined their implications on E1 function with the specific DMMs. We found that the dynamics of phosphorylation Loop A, which is crucial for E1 biological activity, changes with DMMs that are at least about 15 Å away. Because communication is essential for PDC-E1 activity (with alternating active sites), we also investigated the possible communication network within WT PDC-E1 via centrality analysis. We observed that DMMs altered/disrupted the communication network of PDC-E1. Collectively, these results indicate allosteric effect in PDC-E1, with implications for the development of novel small-molecule therapeutics for specific recurrent E1α DMMs such as replacements of R349 responsible for â¼10% of PDC deficiency due to E1α DMMs.
Asunto(s)
Piruvato Deshidrogenasa (Lipoamida) , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa , Humanos , Mitocondrias , Mutación , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/genética , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genéticaRESUMEN
The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC (nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.
Asunto(s)
Núcleo Celular , Complejo Piruvato Deshidrogenasa , Acetilcoenzima A/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Laminas/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Membrana Nuclear/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli.
Asunto(s)
Mitocondrias , Complejo Piruvato Deshidrogenasa , Núcleo Celular/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Pyruvate dehydrogenase complex deficiency is a major cause of primary lactic acidemia resulting in high morbidity and mortality, with limited therapeutic options. PDHA1 mutations are responsible for >82% of cases. The E1 component of PDC is a symmetric dimer of heterodimers (αß/α'ß') encoded by PDHA1 and PDHB. We measured solvent accessibility surface area (SASA), utilized nearest-neighbor analysis, incorporated sequence changes using mutagenesis tool in PyMOL, and performed molecular modeling with SWISS-MODEL, to investigate the impact of residues with disease-causing missense variants (DMVs) on E1 structure and function. We reviewed 166 and 13 genetically resolved cases due to PDHA1 and PDHB, respectively, from variant databases. We expanded on 102 E1α and 13 E1ß nonduplicate DMVs. DMVs of E1α Arg112-Arg224 stretch (exons 5-7) and of E1α Arg residues constituted 40% and 39% of cases, respectively, with invariant Arg349 accounting for 22% of arginine replacements. SASA analysis showed that 86% and 84% of residues with nonduplicate DMVs of E1α and E1ß, respectively, are solvent inaccessible ("buried"). Furthermore, 30% of E1α buried residues with DMVs are deleterious through perturbation of subunit-subunit interface contact (SSIC), with 73% located in the Arg112-Arg224 stretch. E1α Arg349 represented 74% of buried E1α Arg residues involved in SSIC. Structural perturbations resulting from residue replacements in some matched neighboring pairs of amino acids on different subunits involved in SSIC at 2.9-4.0 Å interatomic distance apart, exhibit similar clinical phenotype. Collectively, this work provides insight for future target-based advanced molecular modeling studies, with implications for development of novel therapeutics for specific recurrent DMVs of E1α.
Asunto(s)
Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa , Humanos , Mutación , Mutación Missense , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/genética , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , SolventesRESUMEN
The vast clinical and radiological spectrum of pyruvate dehydrogenase complex (PDHc) deficiency continues to pose challenges both in diagnostics and disease monitoring. Prompt diagnosis is important to enable early initiation of ketogenic diet. The patients were recruited from an ongoing population-based study in Sweden. All patients with a genetically confirmed diagnosis who had been investigated with an MRI of the brain were included. Repeated investigations were assessed to study the evolution of the MRI changes. Sixty-two MRI investigations had been performed in 34 patients (23 females). The genetic cause was mutations in PDHA1 in 29, PDHX and DLAT in 2 each, and PDHB in 1. The lesions were prenatal developmental in 16, prenatal clastic in 18, and postnatal clastic in 15 individuals. Leigh-like lesions with predominant involvement of globus pallidus were present in 12, while leukoencephalopathy was present in 6 and stroke-like lesions in 3 individuals. A combination of prenatal developmental and clastic lesions was present in 15 individuals. In addition, one male with PDHA1 also had postnatal clastic lesions. The most common lesions found in our study were agenesis or hypoplasia of corpus callosum, ventriculomegaly, or Leigh-like lesions. Furthermore, we describe a broad spectrum of other MRI changes that include leukoencephalopathy and stroke-like lesions. We argue that a novel important clue, suggesting the possibility of PDHc deficiency on MRI scans, is the simultaneous presence of multiple lesions on MRI that have occurred during different phases of brain development.
Asunto(s)
Leucoencefalopatías , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa , Accidente Cerebrovascular , Encéfalo/patología , Femenino , Humanos , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/patología , Imagen por Resonancia Magnética , Masculino , Embarazo , Piruvato Deshidrogenasa (Lipoamida)/genética , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by impaired social interaction and behavioural abnormalities. Growing evidence proved that impairment in mitochondrial functions could inhibit energy production and may contribute to the onset of ASD. Genetic variants in the genes of mitochondrial DNA (mtDNA) could interrupt the normal energy metabolism and production in the brain which lead to a wide range of structural and functional changes in the brain resulting in ASD. The present study aims to compare the activities of mitochondrial electron transport chain (ETC) complex I, pyruvate dehydrogenase (PDH) and specific mitochondrial DNA gene (MT-ND1 and MT-ND4) variants associated with ASD subjects in the Tamil Nadu population. Mutational analysis revealed that most mutations in ASD subjects showed synonymous type followed by missense in both the ND1 and ND4 genes. Interestingly, we found that the complex I and PDH dysfunctions may have a role in ASD compared to the controls (p ≤ 0.0001). Hence, the results of the present study suggest that mitochondrial dysfunction, specifically the complex I genes, may play a major role in the onset of ASD, concluding that further research on mitochondrial genes are mandatory to unravel the mechanism behind ASD pathogenesis.
Asunto(s)
Trastorno del Espectro Autista/genética , Complejo I de Transporte de Electrón/genética , Mutación/genética , NADH Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/genética , Adolescente , Trastorno del Espectro Autista/epidemiología , Trastorno del Espectro Autista/metabolismo , Niño , Preescolar , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Activación Enzimática/fisiología , Femenino , Humanos , India/epidemiología , Masculino , Polimorfismo de Nucleótido Simple/genética , Vigilancia de la Población , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
BACKGROUND: Pyruvate dehydrogenase complex (PDHC) deficiency is a common neurodegenerative disease associated with abnormal mitochondrial energy metabolism. The diagnosis of PDHC is difficult because of the lack of a rapid, accurate, and cost-effective clinical diagnostic method. METHODS: A 4-year-old boy was preliminarily diagnosed with putative Leigh syndrome based on the clinical presentation. PDHC activity in peripheral blood leukocytes and a corresponding gene analysis were subsequently undertaken. Sodium pyruvate 1-13 C was used for the analysis of PDHC activity in peripheral leukocytes. The genes encoding PDHC were then scanned for mutations. RESULTS: The results showed that the corresponding PDHC activity was dramatically decreased to 10.5 nmol/h/mg protein as compared with that of healthy controls (124.6 ± 7.1 nmol/h/mg). The ratio of PDHC to citrate synthase was 2.1% (control: 425.3 ± 27.1). The mutation analysis led to the identification of a missense mutation, NM_000284.4:g214C>T, in exon 3 of PDHC. CONCLUSION: The peripheral blood leukocyte PDHC activity assay may provide a practical enzymatic diagnostic method for PDHC-related mitochondrial diseases.
Asunto(s)
Pruebas Enzimáticas Clínicas/métodos , Leucocitos/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/diagnóstico , Complejo Piruvato Deshidrogenasa/metabolismo , Preescolar , Pruebas Genéticas/métodos , Humanos , Masculino , Mutación Missense , Complejo Piruvato Deshidrogenasa/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co-amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI-613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex-associated metabolic vulnerability.
Asunto(s)
Proliferación Celular/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Proteínas de Neoplasias/genética , Complejo Piruvato Deshidrogenasa/genética , Animales , Caprilatos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/terapia , Xenoinjertos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Complejo Piruvato Deshidrogenasa/antagonistas & inhibidores , Complejo Piruvato Deshidrogenasa/metabolismo , Sulfuros/farmacología , Regulación hacia ArribaRESUMEN
Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h-1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.
Asunto(s)
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismo , Acetilcoenzima A/metabolismo , Escherichia coli/genética , L-Lactato Deshidrogenasa/genética , Ingeniería Metabólica , Mutación , Fosfotransferasas (Aceptores Pareados)/genética , Piruvato Oxidasa/genéticaRESUMEN
Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.
Asunto(s)
Infecciones por Mycoplasma , Mycoplasma synoviae , Animales , Escherichia coli/genética , Cobayas , Infecciones por Mycoplasma/veterinaria , Mycoplasma synoviae/genética , Piruvato Deshidrogenasa (Lipoamida)/genética , Complejo Piruvato Deshidrogenasa/genética , Conejos , Proteínas Recombinantes/genéticaRESUMEN
Pyruvate dehydrogenase kinase (PDK) can regulate the catalytic activity of pyruvate decarboxylation oxidation via the mitochondrial pyruvate dehydrogenase complex, and it further links glycolysis with the tricarboxylic acid cycle and ATP generation. This review seeks to elucidate the regulation of PDK activity in different species, mainly mammals, and the role of PDK inhibitors in preventing increased blood glucose, reducing injury caused by myocardial ischemia, and inducing apoptosis of tumor cells. Regulations of PDKs expression or activity represent a very promising approach for treatment of metabolic diseases including diabetes, heart failure, and cancer. The future research and development could be more focused on the biochemical understanding of the diseases, which would help understand the cellular energy metabolism and its regulation by pharmacological effectors of PDKs.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Diabetes Mellitus/metabolismo , Neoplasias/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Animales , Humanos , Complejo Piruvato Deshidrogenasa/antagonistas & inhibidores , Complejo Piruvato Deshidrogenasa/genéticaRESUMEN
Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.
Asunto(s)
Metabolismo Energético , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Fosforilación , Complejo Piruvato Deshidrogenasa/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/genéticaRESUMEN
BACKGROUND: Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. RESULTS: Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). CONCLUSIONS: We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.
Asunto(s)
Ácidos Grasos no Esterificados/biosíntesis , Ingeniería Metabólica , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Citosol/enzimología , NADP/metabolismo , Oxidación-ReducciónRESUMEN
Oviduct flushing is enriched by a wide variety of nutrients that guide the 3-4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.
Asunto(s)
Blastocisto/metabolismo , Medios de Cultivo Condicionados/farmacología , Exosomas/metabolismo , Mitocondrias/metabolismo , Oviductos/metabolismo , Animales , Blastocisto/efectos de los fármacos , Bovinos , Células Cultivadas , Ciclo del Ácido Cítrico , Células Epiteliales/metabolismo , Femenino , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Mitocondrias/efectos de los fármacos , Oviductos/citología , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Ácido Pirúvico/metabolismoRESUMEN
BACKGROUND: The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDC deficiency can be caused by alterations in any of the genes encoding its several subunits. The resulting phenotype, though very heterogeneous, mainly affects the central nervous system. The aim of this study is to describe and discuss the clinical, biochemical and genotypic information from thirteen PDC deficient patients, thus seeking to establish possible genotype-phenotype correlations. RESULTS: The mutational spectrum showed that seven patients carry mutations in the PDHA1 gene encoding the E1α subunit, five patients carry mutations in the PDHX gene encoding the E3 binding protein, and the remaining patient carries mutations in the DLD gene encoding the E3 subunit. These data corroborate earlier reports describing PDHA1 mutations as the predominant cause of PDC deficiency but also reveal a notable prevalence of PDHX mutations among Portuguese patients, most of them carrying what seems to be a private mutation (p.R284X). The biochemical analyses revealed high lactate and pyruvate plasma levels whereas the lactate/pyruvate ratio was below 16; enzymatic activities, when compared to control values, indicated to be independent from the genotype and ranged from 8.5% to 30%, the latter being considered a cut-off value for primary PDC deficiency. Concerning the clinical features, all patients displayed psychomotor retardation/developmental delay, the severity of which seems to correlate with the type and localization of the mutation carried by the patient. The therapeutic options essentially include the administration of a ketogenic diet and supplementation with thiamine, although arginine aspartate intake revealed to be beneficial in some patients. Moreover, in silico analysis of the missense mutations present in this PDC deficient population allowed to envisage the molecular mechanism underlying these pathogenic variants. CONCLUSION: The identification of the disease-causing mutations, together with the functional and structural characterization of the mutant protein variants, allow to obtain an insight on the severity of the clinical phenotype and the selection of the most appropriate therapy.
Asunto(s)
Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa , Humanos , Mutación/genética , Portugal , Piruvato Deshidrogenasa (Lipoamida)/genética , Complejo Piruvato Deshidrogenasa/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genéticaRESUMEN
The transcription factor PdhR has been recognized as the master regulator of the pyruvate catabolism pathway in Escherichia coli, including both NAD-linked oxidative decarboxylation of pyruvate to acetyl-CoA by PDHc (pyruvate dehydrogenase complex) and respiratory electron transport of NADH to oxygen by Ndh-CyoABCD enzymes. To identify the whole set of regulatory targets under the control of pyruvate-sensing PdhR, we performed genomic SELEX (gSELEX) screening in vitro. A total of 35 PdhR-binding sites were identified along the E. coli K-12 genome, including previously identified targets. Possible involvement of PdhR in regulation of the newly identified target genes was analysed in detail by gel shift assay, RT-qPCR and Northern blot analysis. The results indicated the participation of PdhR in positive regulation of fatty acid degradation genes and negative regulation of cell mobility genes. In fact, GC analysis indicated an increase in free fatty acids in the mutant lacking PdhR. We propose that PdhR is a bifunctional global regulator for control of a total of 16-23 targets, including not only the genes involved in central carbon metabolism but also some genes for the surrounding pyruvate-sensing cellular pathways such as fatty acid degradation and flagella formation. The activity of PdhR is controlled by pyruvate, the key node between a wide variety of metabolic pathways, including generation of metabolic energy and cell building blocks.