RESUMO
Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.
Assuntos
ATP Citrato (pro-S)-Liase , Acetatos , Acetilcoenzima A , Linfócitos T CD8-Positivos , Cromatina , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Animais , Cromatina/metabolismo , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , ATP Citrato (pro-S)-Liase/genética , Camundongos , Acetatos/metabolismo , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética , Acetilação , Camundongos Knockout , Citosol/metabolismo , Histonas/metabolismoRESUMO
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.
Assuntos
Acetilcoenzima A , Proteínas de Arabidopsis , Arabidopsis , Núcleo Celular , Etilenos , Regulação da Expressão Gênica de Plantas , Histonas , Complexo Piruvato Desidrogenase , Acetilação , Etilenos/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Complexo Piruvato Desidrogenase/genética , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Acetilcoenzima A/metabolismo , Transcrição Gênica , Mutação , Transdução de Sinais , Receptores de Superfície CelularRESUMO
The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuAâ¢AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5'-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuAâ¢AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuAâ¢AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.
Assuntos
Acetato-CoA Ligase , Acetilcoenzima A , Bacillus subtilis , Proteínas de Bactérias , Lisina , Acetilação , Lisina/metabolismo , Acetilcoenzima A/metabolismo , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/química , Bacillus subtilis/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Monofosfato de Adenosina/metabolismo , OrganofosfatosRESUMO
Lysine acetylation is an evolutionarily conserved protein modification that changes protein functions and plays an essential role in many cellular processes, such as central metabolism, transcriptional regulation, chemotaxis, and pathogen virulence. It can alter DNA binding, enzymatic activity, protein-protein interactions, protein stability, or protein localization. In prokaryotes, lysine acetylation occurs non-enzymatically and by the action of lysine acetyltransferases (KAT). In enzymatic acetylation, KAT transfers the acetyl group from acetyl-CoA (AcCoA) to the lysine side chain. In contrast, acetyl phosphate (AcP) is the acetyl donor of chemical acetylation. Regardless of the acetylation type, the removal of acetyl groups from acetyl lysines occurs only enzymatically by lysine deacetylases (KDAC). KATs are grouped into three main superfamilies based on their catalytic domain sequences and biochemical characteristics of catalysis. Specifically, members of the GNAT are found in eukaryotes and prokaryotes and have a core structural domain architecture. These enzymes can acetylate small molecules, metabolites, peptides, and proteins. This review presents current knowledge of acetylation mechanisms and functional implications in bacterial metabolism, pathogenicity, stress response, translation, and the emerging topic of protein acetylation in the gut microbiome. Additionally, the methods used to elucidate the biological significance of acetylation in bacteria, such as relative quantification and stoichiometry quantification, and the genetic code expansion tool (CGE), are reviewed.
Assuntos
Bactérias , Proteínas de Bactérias , Processamento de Proteína Pós-Traducional , Acetilação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Bactérias/metabolismo , Bactérias/genética , Lisina/metabolismo , Lisina Acetiltransferases/metabolismo , Lisina Acetiltransferases/genética , Acetilcoenzima A/metabolismoRESUMO
BACKGROUND: Biotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16. RESULTS: This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain. CONCLUSIONS: This investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.
Assuntos
Dióxido de Carbono , Cupriavidus necator , Ácidos Graxos , Ácido Succínico , Dióxido de Carbono/metabolismo , Cupriavidus necator/metabolismo , Ácidos Graxos/metabolismo , Ácido Succínico/metabolismo , Acetilcoenzima A/metabolismo , NADP/metabolismoRESUMO
Chemotherapeutic efficacy is seriously impeded by chemoresistance in more than half of hepatocellular carcinoma (HCC) patients. However, the mechanisms involved in chemotherapy-induced upregulation of chemoresistant genes are not fully understood. Here, this study unravels a novel mechanism controlling nuclear acetyl-CoA production to activate the transcription of chemoresistant genes in HCC. NAT10 is upregulated in HCC tissues and its upregulation is correlated with poor prognosis of HCC patients. NAT10 is also upregulated in chemoresistant HCC cells. Targeting NAT10 increases the cytotoxicity of chemotherapy in HCC cells and mouse xenografts. Upon chemotherapy, NAT10 translocates from the nucleolus to the nucleus to activate the transcription of CYP2C9 and PIK3R1. Additionally, nuclear acetyl-CoA is specifically upregulated by NAT10. Mechanistically, NAT10 binds with ACLY in the nucleus and acetylates ACLY at K468 to counteract the SQSTM1-mediated degradation upon chemotherapy. ACLY K468-Ac specifically accumulates in the nucleus and increases nuclear acetyl-CoA production to activate the transcription of CYP2C9 and PIK3R1 through enhancing H3K27ac. Importantly, K468 is required for nuclear localization of ACLY. Significantly, ACLY K468-Ac is upregulated in HCC tissues, and ablation of ACLY K468-Ac sensitizes HCC cells and mouse xenografts to chemotherapy. Collectively, these findings identify NAT10 as a novel chemoresistant driver and the blockage of NAT10-mediated ACLY K468-Ac possesses the potential to attenuate HCC chemoresistance.
Assuntos
Acetilcoenzima A , Carcinoma Hepatocelular , Núcleo Celular , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Acetilcoenzima A/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Acetilação , Camundongos , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Camundongos Nus , Coenzima A Ligases/metabolismo , Coenzima A Ligases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Acetiltransferases N-Terminal/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Camundongos Endogâmicos BALB C , MasculinoRESUMO
Acetyl coenzyme A synthase (ACS) catalyzes the formation and deconstruction of the key biological metabolite, acetyl coenzyme A (acetyl-CoA). The active site of ACS features a {NiNi} cluster bridged to a [Fe4S4]n+ cubane known as the A-cluster. The mechanism by which the A-cluster functions is debated, with few model complexes able to replicate the oxidation states, coordination features, or reactivity proposed in the catalytic cycle. In this work, we isolate the first bimetallic models of two hypothesized intermediates on the paramagnetic pathway of the ACS function. The heteroligated {Ni2+Ni1+} cluster, [K(12-crown-4)2][1], effectively replicates the coordination number and oxidation state of the proposed "Ared" state of the A-cluster. Addition of carbon monoxide to [1]- allows for isolation of a dinuclear {Ni2+Ni1+(CO)} complex, [K(12-crown-2)n][2] (n = 1-2), which bears similarity to the "ANiFeC" enzyme intermediate. Structural and electronic properties of each cluster are elucidated by X-ray diffraction, nuclear magnetic resonance, cyclic voltammetry, and UV/vis and electron paramagnetic resonance spectroscopies, which are supplemented by density functional theory (DFT) calculations. Calculations indicate that the pseudo-T-shaped geometry of the three-coordinate nickel in [1]- is more stable than the Y-conformation by 22 kcal mol-1, and that binding of CO to Ni1+ is barrierless and exergonic by 6 kcal mol-1. UV/vis absorption spectroscopy on [2]- in conjunction with time-dependent DFT calculations indicates that the square-planar nickel site is involved in electron transfer to the CO π*-orbital. Further, we demonstrate that [2]- promotes thioester synthesis in a reaction analogous to the production of acetyl coenzyme A by ACS.
Assuntos
Níquel , Níquel/química , Níquel/metabolismo , Acetato-CoA Ligase/química , Acetato-CoA Ligase/metabolismo , Modelos Moleculares , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Oxirredução , Acetilcoenzima A/metabolismo , Acetilcoenzima A/químicaRESUMO
Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.
Assuntos
Coenzima A-Transferases , Ácido Láctico , Ácido Láctico/química , Coenzima A-Transferases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/química , Poliésteres/química , Acil Coenzima A/metabolismo , Acil Coenzima A/química , Polímeros/química , Acetilcoenzima A/metabolismo , Acetilcoenzima A/químicaRESUMO
Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within â¼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S â N acetyl transfer mechanism.
Assuntos
Lisina , Compostos de Sulfidrila , Lisina/metabolismo , Lisina/química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Acetilação , Cristalinas/metabolismo , Cristalinas/química , Cristalino/metabolismo , Processamento de Proteína Pós-Traducional , Humanos , Acetilcoenzima A/metabolismo , Acetilcoenzima A/químicaRESUMO
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.
Assuntos
Repressão Catabólica , Pseudomonas putida , Complexo Piruvato Desidrogenase , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Pseudomonas putida/enzimologia , Complexo Piruvato Desidrogenase/metabolismo , Complexo Piruvato Desidrogenase/genética , Hidrocarbonetos Aromáticos/metabolismo , Biodegradação Ambiental , Acetilcoenzima A/metabolismo , Ácido Pirúvico/metabolismo , Deleção de Genes , Redes e Vias Metabólicas/genéticaRESUMO
Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.
Assuntos
Acetiltransferases , Microscopia Crioeletrônica , Lisossomos , Mucopolissacaridose III , Mucopolissacaridose III/genética , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/enzimologia , Humanos , Lisossomos/metabolismo , Lisossomos/enzimologia , Acetiltransferases/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Domínio Catalítico , Mutação , Heparitina Sulfato/metabolismo , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química , Modelos Moleculares , Glucosamina/metabolismo , Glucosamina/química , Acetilação , Membranas Intracelulares/metabolismoRESUMO
Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial ß-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.
Assuntos
Acetilcoenzima A , Hepatócitos , Regeneração Hepática , Mitocôndrias Hepáticas , Ácido Pirúvico , Animais , Hepatócitos/metabolismo , Acetilcoenzima A/metabolismo , Camundongos , Ácido Pirúvico/metabolismo , Mitocôndrias Hepáticas/metabolismo , Oxirredução , Proliferação de Células , Ácidos Graxos/metabolismo , Fígado/metabolismo , Transporte de Elétrons , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , MasculinoRESUMO
Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.
Assuntos
Acetilcoenzima A , Núcleo Celular , Cromatina , Drosophila melanogaster , Animais , Acetato-CoA Ligase/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Discos Imaginais/citologia , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Lisina/metabolismo , Oxirredução , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
The solute carrier family 25 member 1 (Slc25a1)-dependent mitochondrial citrate shuttle is responsible for exporting citrate from the mitochondria to the cytoplasm for supporting lipid biosynthesis and protein acetylation. Previous studies on Slc25a1 concentrated on pathological models. However, the importance of Slc25a1 in maintaining metabolic homeostasis under normal nutritional conditions remains poorly understood. Here, we investigated the mechanism of mitochondrial citrate shuttle in maintaining lipid metabolism homeostasis in male Nile tilapia (Oreochromis niloticus). To achieve the objective, we blocked the mitochondrial citrate shuttle by inhibiting Slc25a1 under normal nutritional conditions. Slc25a1 inhibition was established by feeding Nile tilapia with 250 mg/kg 1,2,3-benzenetricarboxylic acid hydrate for 6 weeks or intraperitoneal injecting them with dsRNA to knockdown slc25a1b for 7 days. The Nile tilapia with Slc25a1 inhibition exhibited an obesity-like phenotype accompanied by fat deposition, liver damage and hyperglycemia. Moreover, Slc25a1 inhibition decreased hepatic citrate-derived acetyl-CoA, but increased hepatic triglyceride levels. Furthermore, Slc25a1 inhibition replenished cytoplasmic acetyl-CoA through enhanced acetate pathway, which led to hepatic triglycerides accumulation. However, acetate-derived acetyl-CoA caused by hepatic Slc25a1 inhibition did not activate de novo lipogenesis, but rather modified protein acetylation. In addition, hepatic Slc25a1 inhibition enhanced fatty acids esterification through acetate-derived acetyl-CoA, which increased Lipin1 acetylation and its protein stability. Collectively, our results illustrate that inhibiting mitochondrial citrate shuttle triggers lipid anabolic remodeling and results in lipid accumulation, indicating the importance of mitochondrial citrate shuttle in maintaining lipid metabolism homeostasis.
Assuntos
Ciclídeos , Ácido Cítrico , Metabolismo dos Lipídeos , Fígado , Triglicerídeos , Animais , Triglicerídeos/metabolismo , Fígado/metabolismo , Masculino , Ciclídeos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Cítrico/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Acetilcoenzima A/metabolismoRESUMO
Female willows exhibit greater drought tolerance and benefit more from exogenous acetic acid (AA)-improved drought tolerance than males. However, the potential mechanisms driving these sex-specific responses remain unclear. To comprehensively investigate the sexually dimorphic responsive mechanisms of willows to drought and exogenous AA, here, we performed physiological, proteomic, Lys-acetylproteomic, and transgenic analyses in female and male Salix myrtillacea exposed to drought and AA-applicated drought treatments, focusing on protein abundance and lysine acetylation (LysAc) changes. Drought-tolerant females suffered less drought-induced photosynthetic and oxidative damage, did not activate AA and acetyl-CoA biosynthesis, TCA cycle, fatty acid metabolism, and jasmonic acid signaling as strongly as drought-sensitive males. Exogenous AA caused overaccumulation of endogenous AA and inhibition of acetyl-CoA biosynthesis and utilization in males. However, exogenous AA greatly enhanced acetyl-CoA biosynthesis and utilization and further enhanced drought performance of females, possibly determining that AA improved drought tolerance more in females than in males. Interestingly, overexpression of acetyl-CoA synthetase (ACS) could reprogram fatty acids, increase LysAc levels, and improve drought tolerance, highlighting the involvement of ACS-derived acetyl-CoA in drought responses. In addition, drought and exogenous AA induced sexually dimorphic LysAc associated with histones, transcription factors, and metabolic enzymes in willows. Especially, exogenous AA may greatly improve the photosynthetic capacity of S. myrtillacea males by decreasing LysAc levels and increasing the abundances of photosynthetic proteins. While hyperacetylation in glycolysis, TCA cycle, and fatty acid biosynthesis potentially possibly serve as negative feedback to acclimate acetyl-CoA biosynthesis and utilization in drought-stressed males and AA-applicated females. Thus, acetyl-CoA biosynthesis and utilization determine the sexually dimorphic responses of S. myrtillacea to drought and exogenous AA.
Assuntos
Ácido Acético , Acetilcoenzima A , Secas , Acetilcoenzima A/metabolismo , Ácido Acético/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Acetilação , Estresse FisiológicoRESUMO
Keratinocyte proliferation and differentiation in epidermis are well-controlled and essential for reacting to stimuli such as ultraviolet light. Imbalance between proliferation and differentiation is a characteristic feature of major human skin diseases such as psoriasis and squamous cell carcinoma. However, the effect of keratinocyte metabolism on proliferation and differentiation remains largely elusive. We show here that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) promotes differentiation while inhibits proliferation of keratinocyte and suppresses psoriasis development. FBP1 is identified among the most upregulated genes induced by UVB using transcriptome sequencing and is elevated especially in upper epidermis. Fbp1 heterozygous mice exhibit aberrant epidermis phenotypes with local hyperplasia and dedifferentiation. Loss of FBP1 promotes proliferation and inhibits differentiation of keratinocytes in vitro. Mechanistically, FBP1 loss facilitates glycolysis-mediated acetyl-CoA production, which increases histone H3 acetylation at lysine 9, resulting in enhanced transcription of proliferation genes. We further find that the expression of FBP1 is dramatically reduced in human psoriatic lesions and in skin of mouse imiquimod psoriasis model. Fbp1 deficiency in mice facilitates psoriasis-like skin lesions development through glycolysis and acetyl-CoA production. Collectively, our findings reveal a previously unrecognized role of FBP1 in epidermal homeostasis and provide evidence for FBP1 as a metabolic psoriasis suppressor.
Assuntos
Diferenciação Celular , Proliferação de Células , Frutose-Bifosfatase , Histonas , Queratinócitos , Psoríase , Animais , Humanos , Camundongos , Acetilcoenzima A/metabolismo , Acetilação , Modelos Animais de Doenças , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfatase/genética , Glicólise , Histonas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Psoríase/patologia , Psoríase/metabolismo , Psoríase/genéticaRESUMO
Serpentinization, a geochemical process found on modern and ancient Earth, provides an ultra-reducing environment that can support microbial methanogenesis and acetogenesis. Several groups of archaea, such as the order Methanocellales, are characterized by their ability to produce methane. Here, we generate metagenomic sequences from serpentinized springs in The Cedars, California, and construct a circularized metagenome-assembled genome of a Methanocellales archaeon, termed Met12, that lacks essential methanogenesis genes. The genome includes genes for an acetyl-CoA pathway, but lacks genes encoding methanogenesis enzymes such as methyl-coenzyme M reductase, heterodisulfide reductases and hydrogenases. In situ transcriptomic analyses reveal high expression of a multi-heme c-type cytochrome, and heterologous expression of this protein in a model bacterium demonstrates that it is capable of accepting electrons. Our results suggest that Met12, within the order Methanocellales, is not a methanogen but a CO2-reducing, electron-fueled acetogen without electron bifurcation.
Assuntos
Metano , Metano/metabolismo , Genoma Arqueal , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Metagenoma/genética , Filogenia , Acetilcoenzima A/metabolismo , Dióxido de Carbono/metabolismo , MetagenômicaRESUMO
BACKGROUND: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. RESULTS: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. CONCLUSIONS: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.
Assuntos
Ácido 3-Hidroxibutírico , Acetilcoenzima A , Citrato (si)-Sintase , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Acetilcoenzima A/metabolismo , Citrato (si)-Sintase/metabolismo , Citrato (si)-Sintase/genética , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/biossíntese , Engenharia Metabólica/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cetona Oxirredutases/metabolismo , Cetona Oxirredutases/genética , Oxirredutases do ÁlcoolRESUMO
Driver genomic mutations in tumors define specific molecular subtypes that display distinct malignancy competence, therapeutic resistance and clinical outcome. Although TP53 mutation has been identified as the most common mutation in hepatocellular carcinoma (HCC), current understanding on the biological traits and therapeutic strategies of this subtype has been largely unknown. Here, we reveal that fatty acid ß oxidation (FAO) is remarkable repressed in TP53 mutant HCC and which links to poor prognosis in HCC patients. We further demonstrate that carnitine palmitoyltransferase 1 (CPT1A), the rate-limiting enzyme of FAO, is universally downregulated in liver tumor tissues, and which correlates with poor prognosis in HCC and promotes HCC progression in the de novo liver tumor and xenograft tumor models. Mechanically, hepatic Cpt1a loss disrupts lipid metabolism and acetyl-CoA production. Such reduction in acetyl-CoA reduced histone acetylation and epigenetically reprograms branched-chain amino acids (BCAA) catabolism, and leads to the accumulation of cellular BCAAs and hyperactivation of mTOR signaling. Importantly, we reveal that genetic ablation of CPT1A renders TP53 mutant liver cancer mTOR-addicted and sensitivity to mTOR inhibitor AZD-8055 treatment. Consistently, Cpt1a loss in HCC directs tumor cell therapeutic response to AZD-8055. CONCLUSION: Our results show genetic evidence for CPT1A as a metabolic tumor suppressor in HCC and provide a therapeutic approach for TP53 mutant HCC patients.
Assuntos
Carcinoma Hepatocelular , Carnitina O-Palmitoiltransferase , Neoplasias Hepáticas , Mutação , Proteína Supressora de Tumor p53 , Humanos , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Linhagem Celular Tumoral , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Metabolismo dos Lipídeos/genética , Transdução de Sinais , Acetilcoenzima A/metabolismo , Regulação Neoplásica da Expressão Gênica , MasculinoRESUMO
tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.