RESUMO
The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.
Assuntos
Doença Aguda , Doença Crônica , Genes Bacterianos , Oxigênio , Infecções por Pseudomonas , Pseudomonas aeruginosa , RNA Bacteriano , Animais , Humanos , Oxigênio/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fibrose Cística/microbiologia , Ferimentos e Lesões/microbiologia , Ubiquinona/biossíntese , Anaerobiose , Genes Bacterianos/genética , Sepse/complicações , Sepse/microbiologiaRESUMO
BACKGROUND: Several Rhodobacter sphaeroides have been widely applied in commercial CoQ10 production, but they have poor glucose use. Strategies for enhancing glucose use have been widely exploited in R. sphaeroides. Nevertheless, little research has focused on the role of glucose transmembrane in the improvement of production. RESULTS: There are two potential glucose transmembrane pathways in R. sphaeroides ATCC 17023: the fructose specific-phosphotransferase system (PTSFru, fruAB) and non-PTS that relied on glucokinase (glk). fruAB mutation revealed two effects on bacterial growth: inhibition at the early cultivation phase (12-24 h) and promotion since 36 h. Glucose metabolism showed a corresponding change in characteristic vs. the growth. For ΔfruAΔfruB, maximum biomass (Biomax) was increased by 44.39% and the CoQ10 content was 27.08% more than that of the WT. glk mutation caused a significant decrease in growth and glucose metabolism. Over-expressing a galactose:H+ symporter (galP) in the ΔfruAΔfruB relieved the inhibition and enhanced the growth further. Finally, a mutant with rapid growth and high CoQ10 titer was constructed (ΔfruAΔfruB/tac::galPOP) using several glucose metabolism modifications and was verified by fermentation in 1 L fermenters. CONCLUSIONS: The PTSFru mutation revealed two effects on bacterial growth: inhibition at the early cultivation phase and promotion later. Additionally, biomass yield to glucose (Yb/glc) and CoQ10 synthesis can be promoted using fruAB mutation, and glk plays a key role in glucose metabolism. Strengthening glucose transmembrane via non-PTS improves the productivity of CoQ10 fermentation.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Engenharia Metabólica , Rhodobacter sphaeroides/metabolismo , Ubiquinona/análogos & derivados , Proteínas de Bactérias/genética , Transporte Biológico , Biomassa , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Escherichia coli/genética , Fermentação , Glucoquinase/genética , Glucoquinase/metabolismo , Microbiologia Industrial , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Quinases/genética , Rhodobacter sphaeroides/genética , Ubiquinona/biossínteseRESUMO
Primary coenzyme Q10 (CoQ) deficiency includes a heterogeneous group of mitochondrial diseases characterized by low mitochondrial levels of CoQ due to decreased endogenous biosynthesis rate. These diseases respond to CoQ treatment mainly at the early stages of the disease. The advances in the next generation sequencing (NGS) as whole-exome sequencing (WES) and whole-genome sequencing (WGS) have increased the discoveries of mutations in either gene already described to participate in CoQ biosynthesis or new genes also involved in this pathway. However, these technologies usually provide many mutations in genes whose pathogenic effect must be validated. To functionally validate the impact of gene variations in the disease's onset and progression, different cell models are commonly used. We review here the use of yeast strains for functional complementation of human genes, dermal skin fibroblasts from patients as an excellent tool to demonstrate the biochemical and genetic mechanisms of these diseases and the development of human-induced pluripotent stem cells (hiPSCs) and iPSC-derived organoids for the study of the pathogenesis and treatment approaches.
Assuntos
Ataxia/genética , Ataxia/patologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Debilidade Muscular/genética , Debilidade Muscular/patologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Ataxia/diagnóstico , Exoma/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças Mitocondriais/diagnóstico , Debilidade Muscular/diagnóstico , Ubiquinona/análise , Ubiquinona/biossíntese , Ubiquinona/genética , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Francisella tularensis is the causative agent of tularemia. Because of its extreme infectivity and high mortality rate, this pathogen was classified as a biothreat agent. Francisella spp. are strict aerobes, and ubiquinone (UQ) has been previously identified in these bacteria. While the UQ biosynthetic pathways were extensively studied in Escherichia coli, allowing the identification of 15 Ubi proteins to date, little is known about Francisella spp. In this study, and using Francisella novicida as a surrogate organism, we first identified ubiquinone 8 (UQ8) as the major quinone found in the membranes of this bacterium. Next, we characterized the UQ biosynthetic pathway in F. novicida using a combination of bioinformatics, genetics, and biochemical approaches. Our analysis disclosed the presence in Francisella of 10 putative Ubi proteins, and we confirmed 8 of them by heterologous complementation in E. coli. The UQ biosynthetic pathways from F. novicida and E. coli share similar patterns. However, differences were highlighted: the decarboxylase remains unidentified in Francisella spp., and homologs of the Ubi proteins involved in the O2-independent UQ pathway are not present. This is in agreement with the strictly aerobic niche of this bacterium. Next, via two approaches, i.e., the use of an inhibitor (3-amino-4-hydroxybenzoic acid) and a transposon mutant, both of which strongly impair the synthesis of UQ, we demonstrated that UQ is essential for the growth of F. novicida in respiratory medium and contributes to its pathogenicity in Galleria mellonella used as an alternative animal model. IMPORTANCE Francisella tularensis is the causative bacterium of tularemia and is classified as a biothreat agent. Using multidisciplinary approaches, we investigated the ubiquinone (UQ) biosynthetic pathway that operates in F. novicida used as a surrogate. We show that UQ8 is the major quinone identified in the membranes of Francisella novicida. We identified a new competitive inhibitor that strongly decreased the biosynthesis of UQ. Our demonstration of the crucial roles of UQ for the respiratory metabolism of F. novicida and for the involvement in its pathogenicity in the Galleria mellonella model should stimulate the search for selective inhibitors of bacterial UQ biosynthesis.
Assuntos
Francisella/patogenicidade , Ubiquinona/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Regulação Bacteriana da Expressão Gênica/fisiologia , VirulênciaRESUMO
Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with 18O2 or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with 18O2 to identify the polar oxy-metabolome, defined as polar metabolites labelled with 18O under different physiological O2 tensions. The most highly 18O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies1-4 and cancers with HPDL overexpression5.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Ácidos Mandélicos/metabolismo , Metaboloma , Ubiquinona/análogos & derivados , Animais , Linhagem Celular , Feminino , Humanos , Ácidos Mandélicos/análise , Camundongos , Camundongos Nus , Tirosina/metabolismo , Ubiquinona/biossínteseRESUMO
Methylobacterium sp. CLZ was isolated from soil contaminated with chemical wastewater. This strain simultaneously synthesizes Pyrroloquinoline quinone (PQQ), Coenzyme Q10 (CoQ10), and carotenoids by utilizing methanol as a carbon source. Comparative genomic analysis was performed for five Methylobacterium strains. As per the outcomes, the Methylobacterium CLZ strain showed the smallest genome size and the lowest number of proteins. Thus, it can serve as an ideal cell model for investigating the biological process of Methylobacterium and constructing genetically engineered Methylobacterium. The Methylobacterium CLZ strain's pqqL gene, which does not occur in other Methylobacterium strains but plays a crucial role in PQQ synthesis. This was a surprising finding for the study of PQQ biosynthesis in Methylobacterium. Methylobacterium sp. NI91 strain was generated by random mutagenesis of CLZ strain, and NI91 strain showed a 72.44% increase in PQQ yield. The mutation in the mxaJ gene involved in the methanol dehydrogenase (MDH) synthesis was identified through comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ. The mxaJ gene was found to be upregulated in the NI91 strain. Thus, the up-regulation of the mxaJ gene could be correlated with the high yield of PQQ, and it could provide valuable clues for strain engineering to improve PQQ production.
Assuntos
Proteínas de Bactérias/genética , Genômica/métodos , Methylobacterium/genética , Cofator PQQ/biossíntese , Carotenoides/metabolismo , Regulação Bacteriana da Expressão Gênica , Tamanho do Genoma , Methylobacterium/isolamento & purificação , Methylobacterium/metabolismo , Mutagênese , Microbiologia do Solo , Ubiquinona/análogos & derivados , Ubiquinona/biossíntese , Águas Residuárias/microbiologiaRESUMO
To avoid irreversible stationary phase adsorption and tedious and time-consuming separation steps, high-speed countercurrent chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time, and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as follows: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21°C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol, and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0 mL/min. The yield of isolated antroquinonol was determined to be 6.0 mg from 0.67 g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra-high-performance liquid chromatography-tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed that high-speed countercurrent chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of A. camphorata.
Assuntos
Antineoplásicos/isolamento & purificação , Distribuição Contracorrente/métodos , Polyporales/química , Ubiquinona/análogos & derivados , Extratos Vegetais/química , Ubiquinona/biossíntese , Ubiquinona/isolamento & purificaçãoRESUMO
Mammalian heart development relies on cardiomyocyte mitochondrial maturation and metabolism. Embryonic cardiomyocytes make a metabolic shift from anaerobic glycolysis to oxidative metabolism by mid-gestation. VHL-HIF signaling favors anaerobic glycolysis but this process subsides by E14.5. Meanwhile, oxidative metabolism becomes activated but its regulation is largely elusive. Here, we first pinpointed a crucial temporal window for mitochondrial maturation and metabolic shift, and uncovered the pivotal role of the SRCAP chromatin remodeling complex in these processes in mouse. Disruption of this complex massively suppressed the transcription of key genes required for the tricarboxylic acid cycle, fatty acid ß-oxidation and ubiquinone biosynthesis, and destroyed respirasome stability. Furthermore, we found that the SRCAP complex functioned through H2A.Z deposition to activate transcription of metabolic genes. These findings have unveiled the important physiological functions of the SRCAP complex in regulating mitochondrial maturation and promoting oxidative metabolism during heart development, and shed new light on the transcriptional regulation of ubiquinone biosynthesis.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Complexos Multiproteicos/metabolismo , Fosforilação Oxidativa , Animais , Ácidos Graxos/metabolismo , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Ubiquinona/biossínteseRESUMO
Coenzyme Q10 (CoQ10 ), a strong antioxidant, is used extensively in food, cosmetic and medicine industries. A natural producer, Rhodopseudomonas palustris, was engineered to overproduce CoQ10 . For increasing the CoQ10 content, crtB gene was deleted to block the carotenoid pathway. crtB gene deletion led to 33% improvement of CoQ10 content over the wild type strain. However, it was found that the yield of hopanoids was also increased by competing for the precursors from carotenoid pathway with CoQ10 pathway. To further increase the CoQ10 content, hopanoid pathway was blocked by deleting shc gene, resulting in R. palustris [Δshc, ΔcrtB] to produce 4·7 mg g-1 DCW CoQ10 , which was 1·2 times higher than the CoQ10 content in the wild type strain. The common strategy of co-expression of rate-limiting enzymes (DXS, DPS and UbiA) was combined with the pathway blocking method resulted in 8·2 mg g-1 DCW of CoQ10 , which was 2·9 times higher than that of wild type strain. The results suggested a synergistic effect among different metabolic engineering strategies. This study demonstrates the potential of R. palustris for CoQ10 production and provides viable strategies to increase CoQ10 titer.
Assuntos
Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Rodopseudomonas/enzimologia , Rodopseudomonas/genética , Ubiquinona/análogos & derivados , Carotenoides/metabolismo , Enzimas/genética , Ubiquinona/biossínteseRESUMO
Inside mitochondria reside semi-autonomous genomes, called mtDNA. mtDNA is multi-copy per cell and mtDNA copy number can vary from hundreds to thousands of copies per cell. The variability of mtDNA copy number between tissues, combined with the lack of variability of copy number within a tissue, suggest a homeostatic copy number regulation mechanism. Mutations in the gene encoding the Caenorhabditis elegans hydroxylase, CLK-1, result in elevated mtDNA. CLK-1's canonical role in ubiquinone biosynthesis results in clk-1 mutants lacking ubiquinone. Importantly, clk-1 mutants also exhibit slowed biological timing phenotypes (pharyngeal pumping, defecation, development) and an activated stress response (UPRmt). These biological timing and stress phenotypes have been attributed to ubiquinone deficiency; however, it is unknown whether the mtDNA phenotype is also due to ubiquinone deficiency. To test this, in animals carrying the uncharacterized clk-1 (ok1247) mutant allele, we supplemented with an exogenous ubiquinone precursor 2-4-dihydroxybenzoate (DHB), which has previously been shown to restore ubiquinone biosynthesis. We measured phenotypes as a function of DHB across a log-scale range. Unlike the biological timing and stress phenotypes, the elevated mtDNA phenotype was not rescued. Since CLK-1's canonical role is in ubiquinone biosynthesis and DHB does not rescue mtDNA copy number, we infer CLK-1 has an additional function in homeostatic mtDNA copy number regulation.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Variações do Número de Cópias de DNA , DNA Mitocondrial/metabolismo , Hidroxibenzoatos/farmacologia , Mutação , Ubiquinona/metabolismo , Alelos , Animais , Ubiquinona/biossínteseRESUMO
Coenzyme Q10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome.
Assuntos
Metabolismo Energético , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ubiquinona/análogos & derivados , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Ataxia/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Debilidade Muscular/metabolismo , Nitrobenzoatos/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Ubiquinona/antagonistas & inibidores , Ubiquinona/biossíntese , Ubiquinona/deficiência , Ubiquinona/metabolismoRESUMO
CoenzymeQ10 is one of the main cellular antioxidants and an essential lipid involved in numerous cell reactions, such as energy production and apoptosis modulation. A large number of enzymes are involved in CoQ10 biosynthesis. Mutations in the genes encoding for these enzymes cause a CoQ10 deficiency, characterized by neurological and systemic symptoms. Here we describe two young sisters with sensorineural deafness followed by optic atrophy, due to a novel homozygous pathogenic variant in PDSS1. The visual system seems to be mainly involved when the first steps of CoQ10 synthesis are impaired (PDSS1, PDSS2, and COQ2 deficiency).
Assuntos
Alquil e Aril Transferases/genética , Ataxia/genética , Perda Auditiva Neurossensorial/genética , Doenças Mitocondriais/genética , Debilidade Muscular/genética , Atrofias Ópticas Hereditárias/genética , Ubiquinona/deficiência , Adolescente , Criança , Consanguinidade , Feminino , Humanos , Mutação de Sentido Incorreto , Ubiquinona/análogos & derivados , Ubiquinona/biossíntese , Ubiquinona/genéticaRESUMO
Coenzyme Q (CoQ, ubiquinone) is an essential component of the electron transport system in aerobic organisms. Human type CoQ10, which has 10 units of isoprene in its quinone structure, is especially valuable as a food supplement. Therefore, studying the biosynthesis of CoQ10 is important not only for increasing metabolic knowledge, but also for improving biotechnological production. Herein, we show that Schizosaccharomyces pombe utilizes p-aminobenzoate (PABA) in addition to p-hydroxybenzoate (PHB) as a precursor for CoQ10 synthesis. We explored compounds that affect the synthesis of CoQ10 and found benzoic acid (Bz) at >5 µg/mL inhibited CoQ biosynthesis without accumulation of apparent CoQ intermediates. This inhibition was counteracted by incubation with a 10-fold lower amount of PABA or PHB. Overexpression of PHB-polyprenyl transferase encoded by ppt1 (coq2) also overcame the inhibition of CoQ biosynthesis by Bz. Inhibition by Bz was efficient in S. pombe and Schizosaccharomyces japonicus, but less so in Saccharomyces cerevisiae, Aureobasidium pullulans, and Escherichia coli. Bz also inhibited a S. pombe ppt1 (coq2) deletion strain expressing human COQ2, and this strain also utilized PABA as a precursor of CoQ10. Thus, Bz is likely to inhibit prenylation reactions involving PHB or PABA catalyzed by Coq2.
Assuntos
Ácido Benzoico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/biossíntese , Schizosaccharomyces/metabolismo , Ubiquinona/biossíntese , Proibitinas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Ubiquinona/genéticaRESUMO
Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.
Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supressão Genética , Ubiquinona/biossíntese , Substituição de Aminoácidos , Asparagina , Meios de Cultura/química , Regulação Fúngica da Expressão Gênica , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/genéticaRESUMO
Many proteobacteria, such as Escherichia coli, contain two main types of quinones: benzoquinones, represented by ubiquinone (UQ) and naphthoquinones, such as menaquinone (MK), and dimethyl-menaquinone (DMK). MK and DMK function predominantly in anaerobic respiratory chains, whereas UQ is the major electron carrier in the reduction of dioxygen. However, this division of labor is probably not very strict. Indeed, a pathway that produces UQ under anaerobic conditions in an UbiU-, UbiV-, and UbiT-dependent manner has been discovered recently in E. coli Its physiological relevance is not yet understood, because MK and DMK are also present in E. coli Here, we established that UQ9 is the major quinone of Pseudomonas aeruginosa and is required for growth under anaerobic respiration (i.e. denitrification). We demonstrate that the ORFs PA3911, PA3912, and PA3913, which are homologs of the E. coli ubiT, ubiV, and ubiU genes, respectively, are essential for UQ9 biosynthesis and, thus, for denitrification in P. aeruginosa These three genes here are called ubiTPa , ubiVPa , and ubiUPa We show that UbiVPa accommodates an iron-sulfur [4Fe-4S] cluster. Moreover, we report that UbiUPa and UbiTPa can bind UQ and that the isoprenoid tail of UQ is the structural determinant required for recognition by these two Ubi proteins. Since the denitrification metabolism of P. aeruginosa is believed to be important for the pathogenicity of this bacterium in individuals with cystic fibrosis, our results highlight that the O2-independent UQ biosynthetic pathway may represent a target for antibiotics development to manage P. aeruginosa infections.
Assuntos
Desnitrificação/fisiologia , Pseudomonas aeruginosa/metabolismo , Ubiquinona/biossíntese , Vias Biossintéticas , Respiração Celular , Transporte de Elétrons , Oxigênio/metabolismo , Quinonas/metabolismo , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMO
BACKGROUND: In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms. RESULTS: To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C. CONCLUSION: This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry.
Assuntos
Proteínas Fúngicas/genética , Maleimidas/metabolismo , Polyporales/genética , Polyporales/metabolismo , Ubiquinona/análogos & derivados , Vias Biossintéticas , Fermentação , Proteínas Fúngicas/metabolismo , Polyporales/enzimologia , RNA-Seq , Transcriptoma , Ubiquinona/biossínteseRESUMO
In heart transplantation, time restriction is an unavoidable thorny problem during cardiac transport. Cold storage is an important organ preservation method in donor heart transport. Cold-inducible RNA binding protein (CIRBP) has been proven to play a protective role under cold stress. In this study, we investigated the role of CIRBP in hypothermic cardioprotection during heart preservation in UW solution and explored a new approach to extend the heart preservation time. Cirbp-knockout (Cirbp-/- ), Cirbp-transgenic (Cirbp-Tg), and wild-type rats were, respectively, randomized into two groups based on various heart preservation times (6 or 12-hour group) (n = 8 per group). After preservation in UW solution, all hearts were mounted on a Langendorff apparatus and underwent measurement of cardiac parameters, histological analysis, and molecular study. Within the 6-hour preservation group, no significant difference was found in cardiac functions and histological changes between different rat species. However, after 12 hours of preservation, Cirbp-/- rat hearts showed more apoptosis and worse cardiac function, but less apoptosis and better cardiac function were observed in Cirbp-Tg rat hearts. Furthermore, we found CIRBP-mediated cardiac ubiquinone (CoQ10 ) biosynthesis plays an important role in extending heart preservation, and ubiquinone biosynthesis protein COQ9 was an essential down-stream regulator during this process. Finally, we found that zr17-2, a CIRBP agonist, could enhance the expression of CIRBP, which further enhances the synthesis of CoQ10 and promotes scavenging of reactive oxygen species and ATP production to extend heart preservation. This study demonstrated that CIRBP-enhanced CoQ10 biosynthesis during hypothermic heart preservation and zr17-2-supplemented UW solution could be a promising approach to ameliorate heart damage and extend heart preservation during cardiac transport.
Assuntos
Isquemia Fria/efeitos adversos , Proteínas e Peptídeos de Choque Frio/agonistas , Coração/efeitos dos fármacos , Soluções para Preservação de Órgãos/farmacologia , Preservação de Órgãos/métodos , Proteínas de Ligação a RNA/agonistas , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas e Peptídeos de Choque Frio/genética , Proteínas e Peptídeos de Choque Frio/metabolismo , Técnicas de Inativação de Genes , Transplante de Coração/métodos , Preparação de Coração Isolado , Masculino , Miocárdio/metabolismo , Perfusão/métodos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Transgênicos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/biossínteseRESUMO
PURPOSE: To explore the effects of depression on cardiac autonomic nerve function and related metabolic pathways, the heart rate variability (HRV) and urinary differential metabolites were detected on the college students with depression. METHODS: 12 female freshmen with depression were filtered by the Beck Depression Inventory (BDI-II) and Self-rating Depression Scale (SDS). By wearing an HRV monitoring system, time domain indexes and frequency domain indexes were measured over 24 hours. Liquid chromatography-mass spectrometry (LC-MS) was used to detect their urinary differential metabolites. Differential metabolites were identified by principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). The metabolic pathways related to these differential metabolites were analyzed by the MetPA database. RESULTS: Stress time was significantly increased, and recovery time was markedly decreased in the depression group compared with the control group (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (p < 0.001). Standard deviation of the normal-to-normal R interval (SDNN), root mean square of the beat-to-beat differences (RMSSD), high frequency (HF), and low frequency (LF) were decreased significantly (. CONCLUSION: Some autonomic nervous system disruption, high stress, and poor fatigue recovery were confirmed in college students with depression. The metabolic mechanism involved the disruption of coenzyme Q biosynthesis, glycine-serine-threonine metabolism, tyrosine metabolism, pyrimidine metabolism, and steroid metabolism under daily stress.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Frequência Cardíaca/fisiologia , Ubiquinona/biossíntese , Adolescente , Depressão , Fadiga , Feminino , Glicina/metabolismo , Humanos , Metabolômica , Monitorização Fisiológica , Pirimidinas/metabolismo , Serina/metabolismo , Esteroides/metabolismo , Estresse Fisiológico , Estudantes , Treonina/metabolismo , Tirosina/metabolismo , Ubiquinona/fisiologia , Urina/química , Adulto JovemRESUMO
Coenzyme Q (Q n ) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.
Assuntos
Deleção de Genes , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquinona/análogos & derivados , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biossíntese , Ubiquinona/deficiência , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. PGRPs induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (O2) to hydrogen peroxide (H2O2). In this study we identify the site of PGRP-induced generation of H2O2 in Escherichia coli. Tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (FDH) genes had the highest survival following PGRP treatment. Mutants lacking functional FDH-O had abolished PGRP-induced H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases' substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-I, which results in oxidative stress. These results reveal several essential steps in PGRP-induced bacterial killing.