RESUMO
The adipocyte-derived hormone, leptin, plays a key role in the maintenance of energy homeostasis. Leptin binds to the long form of its receptor, which is predominantly expressed in various hypothalamic regions, including the lateral hypothalamic area (LH) and supraoptic nucleus (SO). Several studies have suggested that leptin directly activates neuronal nitric oxide synthase, leading to increased nitric oxide production. We used histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as a marker for nitric oxide synthase activity and assessed the effect of leptin on nitrergic neurons in the LH and SO of rats. We found that intraperitoneal administration of leptin led to a significant increase in the number of NADPH-d-positive neurons in the LH and SO. In addition, the intensity (optical density) of NADPH-d staining in LH and SO neurons was significantly elevated in rats that received leptin compared with saline-treated rats. These findings suggest that nitrergic neurons in the LH and SO may be implicated in mediating the central effects of leptin.
Assuntos
Região Hipotalâmica Lateral , Leptina , Neurônios Nitrérgicos , Núcleo Supraóptico , Animais , Leptina/farmacologia , Leptina/metabolismo , Masculino , Região Hipotalâmica Lateral/efeitos dos fármacos , Região Hipotalâmica Lateral/metabolismo , Ratos , Núcleo Supraóptico/efeitos dos fármacos , Núcleo Supraóptico/metabolismo , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/metabolismo , NADPH Desidrogenase/metabolismo , Ratos Wistar , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-DawleyRESUMO
(R)-Citronellal is a valuable molecule as the precursor for the industrial synthesis of (-)-menthol, one of the worldwide best-selling compounds in the flavors and fragrances field. However, its biocatalytic production, even from the optically pure substrate (E)-citral, is inherently limited by the activity of Old Yellow Enzyme (OYE). Herein, we rationally designed a different approach to increase the activity of OYE in biocatalytic production. The activity of OYE from Corynebacterium glutamicum (CgOYE) is increased, as well as superior thermal stability and pH tolerance via truncating the different lengths of regions at N-terminal of CgOYE. Next, we converted the truncation mutant N31-CgOYE, a protein involved in proton transfer for the asymmetric hydrogenation of CC bonds, into highly (R)- and (S)-stereoselective enzymes using only three mutations. The mixture of racemic (E/Z)-citral is reduced into the (R)-citronellal with ee and conversion up to 99 % by the mutant of CgOYE, overcoming the problem of the reduction for the mixtures of (E/Z)-citral in biocatalytic reaction. The present work provides a general and effective strategy for improving the activity of OYE, in which the partially conserved histidine residues provide "tunable gating" for the enantioselectivity for both the (R)- and (S)-isomerases.
Assuntos
Aldeídos , NADPH Desidrogenase , Prótons , NADPH Desidrogenase/química , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Monoterpenos AcíclicosRESUMO
2-nitroaniline (2-NA) is an environmental pollutant and has been extensively used as intermediates in organic synthesis. The presence of 2-NA in the environment is not only harmful for aquatic life but also mutagenic for human beings. In this study, we constructed transgenic rice expressing an Old Yellow Enzyme gene, ScOYE3, from Saccharomyces cerevisiae. The ScOYE3 transgenic plants were comprehensively investigated for their biochemical responses to 2-NA treatment and their 2-NA phytoremediation capabilities. Our results showed that the rice seedlings exposed to 2-NA stress, showed growth inhibition and biomass reduction. However, the transgenic plants exhibited strong tolerance to 2-NA stress compared to wild-type plants. Ectopic expression of ScOYE3 could effectively protect transgenic plants against 2-NA damage, which resulted in less reactive oxygen species accumulation in transgenic plants than that in wild-type plants. Our phytoremediation assay revealed that transgenic plants could eliminate more 2-NA from the medium than wild-type plants. Moreover, omics analysis was performed in order to get a deeper insight into the mechanism of ScOYE3-mediated 2-NA transformation in rice. Altogether, the function of ScOYE3 during 2-NA detoxification was characterized for the first time, which serves as strong theoretical support for the phytoremediation potential of 2-NA by Old Yellow Enzyme genes.
Assuntos
Compostos de Anilina , Oryza , Humanos , Oryza/genética , Oryza/metabolismo , Saccharomyces cerevisiae/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Biodegradação Ambiental , Expressão Ectópica do Gene , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Old Yellow Enzymes (OYEs) are flavin-dependent redox enzymes that promote the asymmetric reduction of activated alkenes. Due to the high importance of flavoenzymes in the metabolism of organisms, the interaction between OYEs from the parasites Trypanosoma cruzi and Leishmania braziliensis and three diterpene icetexanes (brussonol and two analogs), were evaluated in the present study, and differences in the binding mechanism and inhibition capacity of these molecules were examined. Although the aforementioned compounds showed poor and negligible activities against T. cruzi and L. braziliensis cells, respectively, the experiments with the purified enzymes indicated that the interaction occurs by divergent mechanisms. Overall, the ligands' inhibitory effect depends on their accessibility to the N5 position of the flavin's isoalloxazine ring. The results also indicated that the OYEs found in both parasites share structural similarities and showed affinities for the diterpene icetexanes in the same range. Nevertheless, the interaction between OYEs and ligands is directed by enthalpy and/or entropy in distinct ways. In conclusion, the binding site of both OYEs exhibits remarkable plasticity, and a large range of different molecules, including that can be substrates and inhibitors, can bind this site. This plasticity should be considered in drug design using OYE as a target.
Assuntos
Doença de Chagas , Leishmania braziliensis , Trypanosoma cruzi , Humanos , NADPH Desidrogenase/química , NADPH Desidrogenase/farmacologia , Doença de Chagas/parasitologia , Flavinas/farmacologiaRESUMO
Acute myeloid leukemia (AML) with CEBPA bZIP in-frame mutations (CEBPAbZIP-inf) is classified within the favorable-risk group by the 2022 European LeukemiaNet (ELN-2022). However, heterogeneous clinical outcomes are still observed in these patients. In this study, we aimed to investigate the mutation profiles and transcriptomic patterns associated with poor outcomes in patients with CEBPAbZIP-inf. One hundred and thirteen CEBPAbZIP-inf patients were identified in a cohort of 887 AML patients homogeneously treated with intensive chemotherapy. Concurrent WT1 or DNMT3A mutations significantly predicted worse survival in AML patients with CEBPAbZIP-inf. RNA-sequencing analysis revealed an enrichment of interferon (IFN) signaling and metabolic pathways in those with a shorter event-free survival (EFS). CEBPAbZIP-inf patients with a shorter EFS had higher expression of IFN-stimulated genes (IRF2, IRF5, OAS2, and IFI35). Genes in mitochondrial complexes I (NDUFA12 and NDUFB6) and V (ATP5PB and ATP5IF1) were overexpressed and were associated with poorer survival, and the results were independently validated in the TARGET AML cohort. In conclusion, concurrent WT1 or DNMT3A mutations and a dysregulated immune and metabolic state were correlated with poor survival in patients with CEBPAbZIP-inf, and upfront allogeneic transplantation may be indicated for better long-term disease control.
Assuntos
Leucemia Mieloide Aguda , Adulto , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Perfilação da Expressão Gênica , Mutação , Intervalo Livre de Progressão , Redes e Vias Metabólicas , Proteínas Estimuladoras de Ligação a CCAAT/genética , NADPH DesidrogenaseRESUMO
Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309â+â5âGâ>âA) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.
Assuntos
Deficiência Intelectual , Atrofia Óptica , Humanos , Masculino , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , NADPH Desidrogenase/metabolismo , Atrofia Óptica/genética , ProteômicaRESUMO
Regulatory complexities in lipogenesis hinder the harmonization of metabolic carbon precursors towards lipid synthesis. Exploring regulatory complexities in lipogenesis, this study identifies NADP+-dependent isocitrate dehydrogenase (IDH) in Tetradesmus obliquus as a key factor. Overexpression IDH in strains ToIDH-1 and ToIDH-2 resulted in a 1.69 and 1.64-fold increase in neutral lipids, respectively, compared to the wild type, with lipid yield reaching 234.56 and 227.17 mg/L. Notably, despite slower growth, the cellular biomass augmented to 790.67 mg/L. Metabolite analysis indicated a shift in carbon precursors from protein to lipid and carbohydrate synthesis. Morphological observations revealed increases in the volume and number of lipid droplets, alongside a change in the fatty acid profile favoring monounsaturated and saturated fatty acids. Furthermore, IDH overexpression enhanced NADPH production and antioxidant activity, thereby further boosting lipid accumulation when combined with salt stress. This study suggests a pathway for improved lipogenesis and algal growth via metabolic engineering.
Assuntos
Antioxidantes , Isocitrato Desidrogenase , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , NADP/metabolismo , NADPH Desidrogenase , Ácidos Graxos , CarbonoRESUMO
Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5-8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, ß-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. KEY POINTS: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2 of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv.
Assuntos
Aspergillus flavus , NADPH Desidrogenase , Aspergillus flavus/metabolismo , NADPH Desidrogenase/metabolismo , Domínio Catalítico , Catálise , AlcenosRESUMO
Many attempts have been made to develop new agents that target EGFR mutants or regulate downstream factors in various cancers. Cell-based screening showed that a natural small molecule, Ertredin, inhibited the growth of EGFRvIII mutant cancer cells. Previous studies have shown that Ertredin effectively inhibits anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII mutant cDNA. However, the underlying mechanism remains unclear. In this study, we investigated the target protein of Ertredin by combining drug affinity-responsive target stability (DARTS) assays with liquid chromatography-mass spectrometry using label-free Ertredin as a bait and HepG2 cell lysates as a proteome pool. NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (NDUFA12) was identified as an Ertredin-binding protein that was responsible for its biological activity. The interaction between NDUFA12 and Ertredin was validated by DARTS and cellular thermal shift assays. In addition, the genetic knockdown of the identified target, NDUFA12, was shown to suppress cell proliferation. NDUFA12 was identified as a biologically relevant target protein of Ertredin that is responsible for its antitumor activity, and these results provide insights into the role of NDUFA12 as a downstream factor in EGFRvIII mutants.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Proteômica/métodos , Proteínas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , NADPH DesidrogenaseRESUMO
We investigated aging-related changes in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the spinal cord of aged dogs. At all levels of the spinal cord examined, NADPH-d activities were observed in neurons and fibers in the superficial dorsal horn (DH), dorsal gray commissure (DGC) and around the central canal (CC). A significant number of NADPH-d positive macro-diameter fibers, termed megaloneurites, were discovered in the sacral spinal cord (S1-S3) segments of aged dogs. The distribution of megaloneurites was characterized from the dorsal root entry zone (DREZ) into the superficial dorsal horn, along the lateral collateral pathway (LCP) to the region of sacral parasympathetic nucleus (SPN), DGC and around the CC, but not in the cervical, thoracic and lumbar segments. Double staining of NADPH-d histochemistry and immunofluorescence showed that NADPH-d positive megaloneurites co-localized with vasoactive intestinal peptide (VIP) immunoreactivity. We believed that megaloneurites may in part represent visceral afferent projections to the SPN and/or DGC. The NADPH-d megaloneurites in the aged sacral spinal cord indicated some anomalous changes in the neurites, which might account for a disturbance in the aging pathway of the autonomic and sensory nerve in the pelvic visceral organs.
Assuntos
NADPH Desidrogenase , Óxido Nítrico Sintase , Cães , Animais , NADPH Desidrogenase/metabolismo , NADP/metabolismo , Óxido Nítrico Sintase/metabolismo , Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , EnvelhecimentoRESUMO
BACKGROUND: The epigenetic mechanisms of asthma remain largely understudied in African Americans and Hispanics/Latinos, two populations disproportionately affected by asthma. We aimed to identify markers, regions and processes with differential patterns of DNA methylation (DNAm) in whole blood by asthma status in ethnically diverse children and youth, and to assess their functional consequences. METHODS: DNAm levels were profiled with the Infinium MethylationEPIC or HumanMethylation450 BeadChip arrays among 1226 African Americans or Hispanics/Latinos and assessed for differential methylation per asthma status at the CpG and region (differentially methylated region (DMR)) level. Novel associations were validated in blood and/or nasal epithelium from ethnically diverse children and youth. The functional and biological implications of the markers identified were investigated by combining epigenomics with transcriptomics from study participants. RESULTS: 128 CpGs and 196 DMRs were differentially methylated after multiple testing corrections, including 92.3% and 92.8% novel associations, respectively. 41 CpGs were replicated in other Hispanics/Latinos, prioritising cg17647904 (NCOR2) and cg16412914 (AXIN1) as asthma DNAm markers. Significant DNAm markers were enriched in previous associations for asthma, fractional exhaled nitric oxide, bacterial infections, immune regulation or eosinophilia. Functional annotation highlighted epigenetically regulated gene networks involved in corticosteroid response, host defence and immune regulation. Several implicated genes are targets for approved or experimental drugs, including TNNC1 and NDUFA12. Many differentially methylated loci previously associated with asthma were validated in our study. CONCLUSIONS: We report novel whole-blood DNAm markers for asthma underlying key processes of the disease pathophysiology and confirm the transferability of previous asthma DNAm associations to ethnically diverse populations.
Assuntos
Asma , Epigenoma , Criança , Humanos , Adolescente , Epigênese Genética , Asma/genética , Metilação de DNA , Perfilação da Expressão Gênica , NADPH Desidrogenase/genéticaRESUMO
The expression of multifunctional proteins can facilitate the setup of a biotechnology process that requires multiple functions absolved by different proteins. Herein the functional and conformational characterization of a formate dehydrogenase-monooxygenase chimera enzyme is presented. The fused enzyme (FDH-PAMO) was prepared by linking the C-terminus of the mutant NADP+-dependent formate dehydrogenase from Pseudomonas sp. 101 (FDH) to the N-terminus of the NADPH-dependent monooxygenase from Thermobifida fusca (PAMO) through a peptide linker of 9 amino acids (ASGGGGSGT) generating a chimera protein of 107,056 Da. The catalytic properties (e.g., kinetic parameters kcat and Km), stability, fluorescence and circular dichroism spectra showed that the so-obtained chimera enzyme FDH-PAMO retains the same functional and conformational properties of the two parental enzymes. Furthermore, SEC chromatographic analysis indicated that, in solution (pH 7.4), FDH-PAMO assembles to tetramers (up to 4.2 %) due to the propensity of FDH and PAMO to form dimers, up to 96.6 % and 6.2 %, respectively. This study provides valuable insights into the structural stability of a thermostable protein (e.g., PAMO) after increasing its size through fusion with another similarly sized thermostable protein (e.g., FDH).
Assuntos
Formiato Desidrogenases , Oxigenases de Função Mista , Oxigenases de Função Mista/química , NADP/metabolismo , Formiato Desidrogenases/química , NADPH Desidrogenase , Pseudomonas/genética , Pseudomonas/metabolismoRESUMO
Crystal structures of Pseudomonas veroniil-arginine dehydrogenase (l-ArgDH), belonging to the µ-crystallin/ornithine cyclodeaminase family, were determined for the enzyme in complex with l-lysine and NADP+ and with l-arginine and NADPH. The main chain coordinates of the P. veroniil-ArgDH monomer showed notable similarity to those of Archaeoglobus fulgidusl-AlaDH, belonging to the same family, and pro-R specificity similar to l-AlaDH for hydride transfer to NADP+ was postulated. However, the residues recognizing the α-amino group of the substrates differed between the two enzymes. Based on a substrate modeling study, it was proposed that in A. fulgidusl-AlaDH, the amino group of l-alanine interacts via a water molecule (W510) with the side chains of Lys41 and Arg52. By contrast, the α-amino group of l-arginine formed hydrogen bonds with the side chains of Thr224 and Asn225 in P. veroniil-ArgDH. Moreover, the guanidino group of l-arginine was fixed into the active site via hydrogen bonds with the side chain of Asp54. Site-directed mutagenesis suggested that Asp54 plays an important role in maintaining high reactivity against the substrate and that Tyr58 and Lys71 play critical roles in enzyme catalysis.
Assuntos
NADPH Desidrogenase , Cristalinas mu , NADP/metabolismo , Sequência de Aminoácidos , Arginina , Sítios de Ligação , Cristalografia por Raios X , Especificidade por SubstratoRESUMO
Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations.
Assuntos
Doença de Alzheimer , Substância Branca , Animais , Humanos , Substância Branca/metabolismo , Acetilcolinesterase/metabolismo , NADP/metabolismo , Calbindinas/metabolismo , Neurônios/metabolismo , Calbindina 2/metabolismo , NADPH Desidrogenase/metabolismo , Doença de Alzheimer/patologia , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Background: The orbital glands, viz. lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia. Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse). Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved.
Assuntos
Bison , Anidrases Carbônicas , Animais , Feminino , Gravidez , Búfalos/metabolismo , Monofenol Mono-Oxigenase , Fosfatase Alcalina/metabolismo , Bison/metabolismo , NADPH Desidrogenase , L-Lactato Desidrogenase , Di-Hidrolipoamida Desidrogenase , Feto/metabolismo , Di-HidroxifenilalaninaRESUMO
Leber hereditary optic neuropathy (LHON) is a primary inherited neurodegenerative disorder of the optic nerve. It has been ascribed to variants in the mitochondrial genome, mainly the m.3460G>A, m.11778G>A and m.14484T>C mutations in ND1, ND4 and ND6, respectively. Nonetheless, inconclusive molecular diagnosis is not uncommon. Recently, biallelic mutations in the NDUFS2, DNAJC30, MCAT and NDUFA12 nuclear genes have been identified in unresolved LHON cases, identifying an autosomal recessive LHON (arLHON, OMIM:619382). The clinical presentation of arLHON copies that of typical LHON due to mtDNA mutations (mtLHON), with an acute phase of sudden and severe vision loss, telangiectatic and tortuous vessels around the optic nerve and swelling of the retinal nerve fibre layer. This is followed by a chronic phase of retinal nerve fibre layer loss, but eventually affected individuals recover partial or full visual acuity. Idebenone treatment significantly improved vision recovery in DNAJC30-associated patients. As for mtLHON, arLHON predominantly affected male compared with female carriers. The discovery of arLHON cases breaks with the dogma of exclusive maternal inheritance. It defines a new neuro-ophthalmo-genetic paradigm, which should be considered in individuals manifesting a LHON phenotype but with an inconclusive molecular diagnosis. NDUFS2, DNAJC30, MCAT and NDUFA12 should be investigated in these individuals, knowing that other arLHON genes might exist.
Assuntos
Atrofia Óptica Hereditária de Leber , Masculino , Feminino , Humanos , Atrofia Óptica Hereditária de Leber/genética , DNA Mitocondrial , Mutação/genética , Nervo Óptico , Retina , NADPH Desidrogenase/genéticaRESUMO
The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.
Assuntos
Aminoácidos , NADPH Desidrogenase , NADPH Desidrogenase/química , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Monoterpenos Cicloexânicos , Catálise , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.
Assuntos
Mycobacterium smegmatis , Oxirredutases , Oxirredutases/metabolismo , Mycobacterium smegmatis/metabolismo , Oxirredução , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismoRESUMO
Asymmetric reduction by ene-reductases has received considerable attention in recent decades. While several enzyme families possess ene-reductase activity, the Old Yellow Enzyme (OYE) family has received the most scientific and industrial attention. However, there is a limited substrate range and few stereocomplementary pairs of current ene-reductases, necessitating the development of a complementary class. Flavin/deazaflavin oxidoreductases (FDORs) that use the uncommon cofactor F420 have recently gained attention as ene-reductases for use in biocatalysis due to their stereocomplementarity with OYEs. Although the enzymes of the FDOR-As sub-group have been characterized in this context and reported to catalyse ene-reductions enantioselectively, enzymes from the similarly large, but more diverse, FDOR-B sub-group have not been investigated in this context. In this study, we investigated the activity of eight FDOR-B enzymes distributed across this sub-group, evaluating their specific activity, kinetic properties, and stereoselectivity against α,ß-unsaturated compounds. The stereochemical outcomes of the FDOR-Bs are compared with enzymes of the FDOR-A sub-group and OYE family. Computational modelling and induced-fit docking are used to rationalize the observed catalytic behaviour and proposed a catalytic mechanism.
Assuntos
Mycobacterium smegmatis , Oxirredutases , Oxirredutases/metabolismo , Riboflavina/metabolismo , NADPH Desidrogenase/química , Biocatálise , OxirreduçãoRESUMO
Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-ß/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-ß, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-ß/Smad3 pathway.