RESUMO
The anaerobic oxidation of alkanes is a microbial process that mitigates the flux of hydrocarbon seeps into the oceans. In marine archaea, the process depends on sulphate-reducing bacterial partners to exhaust electrons, and it is generally assumed that the archaeal CO2-forming enzymes (CO dehydrogenase and formylmethanofuran dehydrogenase) are coupled to ferredoxin reduction. Here, we study the molecular basis of the CO2-generating steps of anaerobic ethane oxidation by characterising native enzymes of the thermophile Candidatus Ethanoperedens thermophilum obtained from microbial enrichment. We perform biochemical assays and solve crystal structures of the CO dehydrogenase and formylmethanofuran dehydrogenase complexes, showing that both enzymes deliver electrons to the F420 cofactor. Both multi-metalloenzyme harbour electronic bridges connecting CO and formylmethanofuran oxidation centres to a bound flavin-dependent F420 reductase. Accordingly, both systems exhibit robust coupled F420-reductase activities, which are not detected in the cell extract of related methanogens and anaerobic methane oxidisers. Based on the crystal structures, enzymatic activities, and metagenome mining, we propose a model in which the catabolic oxidising steps would wire electron delivery to F420 in this organism. Via this specific adaptation, the indirect electron transfer from reduced F420 to the sulphate-reducing partner would fuel energy conservation and represent the driving force of ethanotrophy.
Assuntos
Dióxido de Carbono , Etano , Oxirredução , Dióxido de Carbono/metabolismo , Etano/metabolismo , Etano/química , Archaea/metabolismo , Archaea/genética , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/química , Cristalografia por Raios X , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/química , Anaerobiose , Ferredoxinas/metabolismo , Riboflavina/análogos & derivadosRESUMO
Approximately two-thirds of the estimated one-billion metric tons of methane produced annually by methanogens is derived from the cleavage of acetate. Acetate is broken down by a Ni-Fe-S-containing A-cluster within the enzyme acetyl-CoA synthase (ACS) to carbon monoxide (CO) and a methyl group (CH3+). The methyl group ultimately forms the greenhouse gas methane, whereas CO is converted to the greenhouse gas carbon dioxide (CO2) by a Ni-Fe-S-containing C-cluster within the enzyme carbon monoxide dehydrogenase (CODH). Although structures have been solved of CODH/ACS from acetogens, which use these enzymes to make acetate from CO2, no structure of a CODH/ACS from a methanogen has been reported. In this work, we use cryo-electron microscopy to reveal the structure of a methanogenic CODH and CODH/ACS from Methanosarcina thermophila (MetCODH/ACS). We find that the N-terminal domain of acetogenic ACS, which is missing in all methanogens, is replaced by a domain of CODH. This CODH domain provides a channel for CO to travel between the two catalytic Ni-Fe-S clusters. It generates the binding surface for ACS and creates a remarkably similar CO alcove above the A-cluster using residues from CODH rather than ACS. Comparison of our MetCODH/ACS structure with our MetCODH structure reveals a molecular mechanism to restrict gas flow from the CO channel when ACS departs, preventing CO escape into the cell. Overall, these long-awaited structures of a methanogenic CODH/ACS reveal striking functional similarities to their acetogenic counterparts despite a substantial difference in domain organization.
Assuntos
Acetato-CoA Ligase , Aldeído Oxirredutases , Microscopia Crioeletrônica , Metano , Methanosarcina , Complexos Multienzimáticos , Aldeído Oxirredutases/metabolismo , Aldeído Oxirredutases/química , Microscopia Crioeletrônica/métodos , Methanosarcina/enzimologia , Methanosarcina/metabolismo , Metano/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/química , Acetato-CoA Ligase/genética , Monóxido de Carbono/metabolismo , Modelos MolecularesRESUMO
Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.
Assuntos
Glioblastoma , Metaboloma , NAD , Glioblastoma/metabolismo , Glioblastoma/patologia , NAD/metabolismo , Humanos , Linhagem Celular Tumoral , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Complexos Multienzimáticos/metabolismo , Levilactobacillus brevis/metabolismo , Invasividade Neoplásica , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Movimento Celular , Trifosfato de Adenosina/metabolismo , NADH NADPH OxirredutasesRESUMO
Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
Assuntos
Metionina , Chaperonas Moleculares , Complexos Multienzimáticos , Processamento de Proteína Pós-Traducional , Ribossomos , Animais , Humanos , Acetilação , Domínio Catalítico , Metionil Aminopeptidases/química , Metionil Aminopeptidases/metabolismo , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Acetiltransferase N-Terminal A/química , Acetiltransferase N-Terminal A/metabolismo , Ribossomos/química , Ribossomos/enzimologia , Ribossomos/metabolismo , Metionina/química , Metionina/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Caenorhabditis elegansRESUMO
2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.
Assuntos
Oxirredutases do Álcool , Butileno Glicóis , Etanol , Butileno Glicóis/metabolismo , Butileno Glicóis/química , Etanol/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , NADH NADPH Oxirredutases/metabolismo , NADH NADPH Oxirredutases/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/químicaRESUMO
Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.
Assuntos
Período de Replicação do DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Instabilidade Genômica , Animais , Feminino , Masculino , Camundongos , Blastocisto/citologia , Blastocisto/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Segregação de Cromossomos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Período de Replicação do DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Fase S/efeitos dos fármacos , Fase S/genética , Análise de Célula Única , Pontos de Quebra do Cromossomo , Divisão Celular , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismoRESUMO
Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5â Å resolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.
Assuntos
Aldose-Cetose Isomerases , Fosfomicina , Complexos Multienzimáticos , Toxoplasma , Toxoplasma/enzimologia , Toxoplasma/efeitos dos fármacos , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Fosfomicina/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografia por Raios X , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/metabolismoRESUMO
DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.
Assuntos
Ciclo Celular , Proteínas Culina , Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Saccharomyces cerevisiae , Adutos de DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Transporte Proteico/genética , Ubiquitinação/genética , Replicação do DNA/genética , Complexos Multienzimáticos/metabolismoRESUMO
There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform. The primary objective of this study was to determine the protective potential of M2e proteins expressed on Lumazine synthase (LS) nanoparticles. M2e-LS proteins, produced through the E. coli system, spontaneously assemble into nanoparticles. The study investigated the efficacy of the M2e-LS nanoparticle vaccine in mice. Mice immunized with M2e-LS nanoparticles exhibited significantly higher levels of intracellular cytokines than those receiving soluble M2e proteins. The M2e-LS protein exhibited robust immunogenicity and provided 100% protection against cross-clade influenza.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza , Complexos Multienzimáticos , Nanopartículas , Infecções por Orthomyxoviridae , Proteínas da Matriz Viral , Animais , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Vírus da Influenza A Subtipo H1N1/imunologia , Nanopartículas/química , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Camundongos , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , Feminino , Camundongos Endogâmicos BALB C , Anticorpos Antivirais/imunologia , Citocinas/metabolismo , Proteção Cruzada/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/imunologia , Influenza Humana/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas ViroporinasRESUMO
Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal-organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD+) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe3O4/NAD+ (ZIF-8@Fe3O4/NAD+) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe3O4/NAD+ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe3O4/NAD+ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe3O4/NAD+ENF after ten cycles. The magnetic metal-organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications.
Assuntos
Acetoína , Biomassa , Etanol , Estruturas Metalorgânicas , Etanol/metabolismo , Etanol/química , Estruturas Metalorgânicas/química , Acetoína/metabolismo , NAD/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Biocombustíveis , Álcool Desidrogenase/metabolismo , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/químicaRESUMO
Dihydro-nicotinamide adenine dinucleotide (NADH) detection is crucial since it is a vital coenzyme in organism metabolism. Compared to the traditional method based on natural NADH oxidase (NOX), nanozymes with multienzyme-like activity can catalyze multistage reactions in a singular setup, simplifying detection processes and enhancing sensitivity. In this study, an innovative NADH detection method was developed using iron-doped carbon (Fe@C) nanozyme synthesized from metal-organic frameworks with in situ reduced Pt clusters. This nanozyme composite (Pt/Fe@C) demonstrated dual NOX and peroxidase-like characteristics, significantly enhancing the catalytic efficiency and enabling NADH conversion to NAD+ and H2O2 with subsequent detection. The collaborative research involving both experimental and theoretical simulations has uncovered the catalytic process and the cooperative effect of Fe and Pt atoms, leading to enhanced oxygen adsorption and activation, as well as a decrease in the energy barrier of the key step in the H2O2 decomposition process. These findings indicate that the catalytic performance of Pt/Fe@C in NOX-like and POD-like reactions can be significantly improved. The colorimetric sensor detects NADH with a limit of detection as low as 0.4 nM, signifying a breakthrough in enzyme-mimicking nanozyme technology for precise NADH measurement.
Assuntos
Carbono , Estruturas Metalorgânicas , NAD , Platina , NAD/química , Estruturas Metalorgânicas/química , Platina/química , Carbono/química , Ferro/química , Peróxido de Hidrogênio/química , Colorimetria/métodos , Humanos , Catálise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Materiais Biomiméticos/química , Limite de Detecção , NADH NADPH OxirredutasesRESUMO
NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 µg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.
Assuntos
Colorimetria , NAD(P)H Desidrogenase (Quinona) , NADH NADPH Oxirredutases , NAD(P)H Desidrogenase (Quinona)/metabolismo , NAD(P)H Desidrogenase (Quinona)/química , Humanos , NADH NADPH Oxirredutases/metabolismo , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Cobalto/química , Carbono/química , Biomimética , Limite de Detecção , Nitrogênio/química , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Ferrocianetos/química , NAD/metabolismo , NAD/químicaRESUMO
In this study, marine medaka (Oryzias melastigma) embryos were exposed to different concentrations of water-accommodated fractions (WAFs) and chemically enhanced water-accommodated fractions (CEWAFs) of Oman crude oil for 14 d by semi-static exposure methods. The effects on growth and development and energy metabolism process were evaluated. Results showed that embryo survival and hatchability were decreased in a dose-dependent manner with an increase in the concentration of petroleum hydrocarbon compounds, whereas the malformation exhibited a dose-dependent increase. Compared to the control, the adenosine triphosphate (ATP) content and Na+-K+-ATPase (NKA) activities of embryos exposed to both WAFs and CEWAFs were reduced, while intracellular reactive oxygen species (ROS) levels and NADH oxidase (NOX) activities were increased. Our study demonstrated that exposure to crude oil dispersed by chemical dispersant affected the growth and development of marine medaka embryos, caused oxidative stress while produced a series of malformations in the body and dysregulation in energy metabolism. In comparison, the toxic effects of chemically dispersed crude oil might be more severe than the oil itself in the equivalent diluted concentration treatment solution. These would provide more valuable and reliable reference data for the use of chemical dispersants in oil spills.
Assuntos
Embrião não Mamífero , Metabolismo Energético , Oryzias , Estresse Oxidativo , Petróleo , Espécies Reativas de Oxigênio , Poluentes Químicos da Água , Animais , Oryzias/metabolismo , Oryzias/embriologia , Petróleo/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Tensoativos/toxicidade , NADH NADPH Oxirredutases/metabolismo , Água/química , Trifosfato de Adenosina/metabolismo , Complexos Multienzimáticos/metabolismoRESUMO
Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.
Assuntos
Modelos Animais de Doenças , Fígado , Camundongos Knockout , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/metabolismo , Fígado/metabolismo , Miopatias Distais/genética , Miopatias Distais/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Ácido N-Acetilneuramínico/metabolismoRESUMO
Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of ß-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.
Assuntos
Autofagia , Cálcio , Miopatias Distais , Proteínas de Choque Térmico HSP70 , Complexos Multienzimáticos , Mutação , Humanos , Autofagia/genética , Autofagia/efeitos dos fármacos , Mutação/genética , Cálcio/metabolismo , Miopatias Distais/genética , Miopatias Distais/metabolismo , Miopatias Distais/patologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Células HEK293 , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , ÍndiaRESUMO
The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.
Assuntos
Endorribonucleases , Complexos Multienzimáticos , Polirribonucleotídeo Nucleotidiltransferase , Porinas , RNA Helicases , Estabilidade de RNA , RNA Mensageiro , Endorribonucleases/metabolismo , Endorribonucleases/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Estabilidade de RNA/genética , Porinas/metabolismo , Porinas/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Pequeno RNA não Traduzido/metabolismo , Pequeno RNA não Traduzido/genética , Regulação Bacteriana da Expressão GênicaRESUMO
A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %. Amination reduced the ß-glucosidase activity of naringinase complex; however, did not affect optimal pH and temperature of the enzyme and it favored immobilization, obtaining α-l-rhamnosidase and ß-d-glucosidase activities of 1.7 and 4.2 times the values obtained when the unmodified enzymes were immobilized. Amination favored the stability of the immobilized biocatalyst, retaining 100 % of both activities after 190 h at 30 °C and pH 3, while its non-aminated counterpart retained 80 and 52 % of α-rhamnosidase and ß-glucosidase activities, respectively. The immobilized catalyst showed a better performance in grapefruit juice debittering, obtaining a naringin conversion of 7 times the value obtained with the non-aminated catalyst.
Assuntos
Enzimas Imobilizadas , Sucos de Frutas e Vegetais , Glioxilatos , Sefarose , Sucos de Frutas e Vegetais/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Aminação , Concentração de Íons de Hidrogênio , Sefarose/química , Glioxilatos/química , Citrus/química , Citrus/enzimologia , Estabilidade Enzimática , Biocatálise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Temperatura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavanonas/química , Flavanonas/metabolismo , CatáliseRESUMO
Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.
Assuntos
Biocatálise , Sistema Livre de Células , Estireno/metabolismo , Estireno/química , Escherichia coli/genética , Escherichia coli/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismoRESUMO
Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.
Assuntos
Cobamidas , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , RNA Mensageiro , Riboswitch , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Cobamidas/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Iniciação Traducional da Cadeia Peptídica , RNA Helicases/genética , RNA Helicases/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas da Membrana Bacteriana Externa , Polirribonucleotídeo Nucleotidiltransferase , Proteínas de Membrana TransportadorasRESUMO
Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, as a potential drug target against Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We employ a high-throughput molecular docking approach to screen ~ 600,000 compounds targeting RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 compounds that exhibit potent antimycobacterial activity. These compounds also reduce intracellular burden of M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these three compounds enhance the bactericidal effect of first-line anti-TB drugs, isoniazid and rifampicin. Corroborating with the in silico predicted high docking scores along with favourable ADME and toxicity profiles, all three compounds demonstrate binding affinity towards purified lumazine synthase enzyme in vitro, in addition these compounds exhibit riboflavin displacement in an in vitro assay with purified lumazine synthase indicative of specificity of these compounds to the active site. Further, treatment of M. tb with these compounds indicate reduced production of flavin adenine dinucleotide (FAD), the ultimate end product of the riboflavin biosynthetic pathway suggesting the action of these drugs on riboflavin biosynthesis. These compounds also show acceptable safety profile in mammalian cells, with a high selective index. Hence, our study validates RibH as an important drug target against M. tb and identifies potent antimycobacterial agents.