RESUMO
Ferroptotic cancer therapy has been extensively investigated since the genesis of the ferroptosis concept. However, the therapeutic efficacy of ferroptosis induction in heterogeneous and plastic melanoma has been compromised, because the melanocytic and transitory cell subpopulation is resistant to iron-dependent oxidative stress. Here, we report a phenotype-altering liposomal nanomedicine to enable the ferroptosis-resistant subtypes of melanoma cells vulnerable to lipid peroxidation via senescence induction. The strategy involves the ratiometric coencapsulation of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor (palbociclib) and a ferroptosis inducer (auranofin) within cRGD peptide-modified targeted liposomes. The two drugs showed a synergistic anticancer effect in the model B16F10 melanoma cells, as evidenced by the combination index analysis (<1). The liposomes could efficiently deliver both drugs into B16F10 cells in a targeted manner. Afterward, the liposomes potently induced the intracellular redox imbalance and lipid peroxidation. Palbociclib significantly provoked cell cycle arrest at the G0/G1 phase, which sensitized auranofin-caused ferroptosis through senescence induction. Meanwhile, palbociclib depleted intracellular glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), further boosting ferroptosis. The proof-of-concept was also demonstrated in the B16F10 tumor-bearing mice model. The current work offers a promising ferroptosis-targeting strategy for effectively treating heterogeneous melanoma by manipulating the cellular plasticity.
Assuntos
Ferroptose , Melanoma , Animais , Camundongos , Melanoma/tratamento farmacológico , Lipossomos/farmacologia , Coenzimas/farmacologia , Auranofina/farmacologia , Peroxidação de LipídeosRESUMO
The incidence of intra-abdominal candidiasis (IAC), characterized by high morbidity and mortality, has become a serious concern. The limitations of current antifungal drugs on the market underscores the importance of the development of novel antifungal agents. In the present study, the antifungal activity of vitamin D3 (VD3) against various Candida species was investigated. In vitro, the broth microdilution method and solid plate assay confirmed that VD3 inhibited the growth of Candida spp. in a broad-spectrum, dose-dependent manner. VD3 also had a significant antifungal effect on the initiation, development, and maturation phases of biofilm formation in Candida albicans. The mechanism of VD3 action was explored by transcriptomics and reverse transcription quantitative PCR (RT-qPCR) analysis, and showed that VD3 affects ribosome biogenesis, coenzyme metabolism, and carbon metabolism. These results suggested that VD3 may have multitarget effects against C. albicans. In the murine IAC model, VD3 reduced the fungal burden in the liver, kidneys, and small intestine. Further histopathological analysis and quantification of plasma cytokine levels confirmed that VD3 treatment significantly decreased the infiltration of inflammatory cells and the levels of plasma interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Taken together, these findings suggest a new antifungal mechanism for VD3 and indicate that VD3 could be an effective therapeutic agent for use in IAC treatment.
Assuntos
Candida albicans , Candidíase , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Carbono , Colecalciferol/farmacologia , Coenzimas/farmacologia , Citocinas , Interferons/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Necrose Tumoral/farmacologiaRESUMO
Purpose: Male infertility is a global public health issue recognized by the WHO. Recently, antioxidants are increasingly used to treat idiopathic male infertility. However, the lack of available evidence has led to the inability to rank the effects of antioxidants on the sperm quality parameters and pregnancy rate of infertile men. This network meta-analysis studied the effects of different antioxidants on the sperm quality and pregnancy rate of idiopathic male infertility. Methods: We searched PubMed, Embase, Web of Science, and Cochrane Library databases for randomized controlled trials (RCTs). The weighted mean difference (WMD) and odds ratio (OR) were applied for the comparison of continuous and dichotomous variables, respectively, with 95% CIs. The outcomes were sperm motility, sperm concentration, sperm morphology, and pregnancy rate. Results: A total of 23 RCTs with 1,917 patients and 10 kids of antioxidants were included. l-Carnitine, l-carnitine+l-acetylcarnitine, coenzyme-Q10, ω-3 fatty acid, and selenium were more efficacious than placebo in sperm quality parameters. l-Carnitine was ranked first in sperm motility and sperm morphology (WMD 6.52% [95% CI: 2.55% to 10.05%], WMD 4.96% [0.20% to 9.73%]). ω-3 fatty acid was ranked first in sperm concentration (WMD 9.89 × 106/ml, [95% CI: 7.01 to 12.77 × 106/ml]). In terms of pregnancy rate, there was no significant effect as compared with placebo. Conclusions: l-Carnitine was ranked first in sperm motility and sperm morphology. ω-3 fatty acid was ranked first in sperm concentration. Coenzyme-Q10 had better effective treatment on sperm motility and concentration. Furthermore, high-quality RCTs with adequate sample sizes should be conducted to compare the outcomes of different antioxidants.
Assuntos
Ácidos Graxos Ômega-3 , Infertilidade Masculina , Antioxidantes/farmacologia , Carnitina , Coenzimas/farmacologia , Coenzimas/uso terapêutico , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-3/uso terapêutico , Feminino , Humanos , Infertilidade Masculina/tratamento farmacológico , Masculino , Metanálise em Rede , Gravidez , Taxa de Gravidez , Ensaios Clínicos Controlados Aleatórios como Assunto , EspermatozoidesRESUMO
Evidences have shown that alterations in testicular dehydrogenase and ionic-ATPase activities have important implications in spermatogenesis and sperm capacitation, a penultimate biochemical change required for fertilization. Previous studies have revealed that taurine and coenzyme-Q10 (COQ-10), which are synergistic testicle-active bioflavonoids, with proven gonadotropin-enhancing properties reduce testicular damage in rats. Hence, this study investigated the effects of taurine and COQ-10 or their combination alone, and in the preventive and reversal of chlorpromazine-induced inhibition of testicular dehydrogenase enzymes, electrogenic pumps, sperm capacitation and acrosomal-reaction in male Wister rats. In the drug-treatment alone or preventive-protocol, rats received oral treatment of saline (10â¯mL/kg), taurine (150â¯mg/kg/day), COQ-10 (10â¯mg/kg/day) or both alone repeatedly for 56 days, or in combination with chlorpromazine (30â¯mg/kg/p.o./day) from days 29-56. In the reversal-protocol, the animals received chlorpromazine for 56 days prior to saline, taurine, COQ-10 or the combination from days 29-56. Thereafter, spermatogenesis (sperm count, viability, motility and morphology), testicular dehydrogenase [3beta-hydroxysteroid dehydrogenase (3ß-HSD), 17beta-hydroxysteroid dehydrogenase (17ß-HSD), glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase-X (LDH-X)], ATPase (Na+/K+, Ca2+, Mg2+, H+) activities, sperm capacitation and acrosomal reaction were evaluated. Taurine and COQ-10 or their combination increased spermatogenesis, testicular 3ß-HSD, 17ß-HSD, G6PDH and LDH-X enzymes of naïve and chlorpromazine-treated rats. Both taurine and COQ-10 increased Na+/K+, Ca2+, Mg2+ and H+-ATPase activities. Also, taurine and COQ-10 or their combination prevented and reversed chlorpromazine-induced inhibition of sperm capacitation and acrosomal-reaction. The study showed that taurine and COQ-10 prevent and reverse chlorpromazine-induced inhibition of spermatogenesis, epididymal sperm capacitation and acrosomal reaction in rats through increased testicular dehydrogenases and electrogenic pump activities.
Assuntos
Antipsicóticos/toxicidade , Clorpromazina/toxicidade , Coenzimas/uso terapêutico , Flavonoides/uso terapêutico , Substâncias Protetoras/uso terapêutico , Taurina/uso terapêutico , Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Coenzimas/farmacologia , Sinergismo Farmacológico , Flavonoides/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Taurina/farmacologia , Testículo/metabolismoRESUMO
Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone) has been reported to exert anticancer properties against human breast/lung cancer cells. This study investigated the in vitro and in vivo anticancer properties of CoQ0 on human ovarian carcinoma (SKOV-3) cells and xenografted nude mice, and revealed the underlying molecular mechanism. CoQ0 induced G2/M arrest through downregulation of cyclin B1/A and CDK1/K2 expressions. CoQ0-induced autophagy as a survival mechanism was evidenced by increased accumulation of LC3-II, GFP-LC3 puncta, AVOs formation and Beclin-1/Bcl-2 dysregulation. Increased TUNEL-positive cells and Annexin-V/PI stained cells indicated CoQ0-induced late apoptosis. Both mitochondrial (caspase-3, PARP and Bax/Bcl-2 dysregulation) and ER stress (caspase-12 and Hsp70) signals are involved in execution of apoptosis. Interestingly, CoQ0-induced apoptosis/autophagy is associated with suppression of HER-2/neu and PI3K/AKT signalling cascades. CoQ0 triggered intracellular ROS production, whereas antioxidant N-acetylcysteine prevented CoQ0-induced apoptosis, but not autophagy. Inhibition of apoptosis by Z-VAD-FMK suppressed CoQ0-induced autophagy (diminished LC3-II/AVOs), indicates CoQ0-induced apoptosis led to evoke autophagy. Contrary, inhibition of autophagy by 3-MA/CQ potentiated CoQ0-induced apoptosis (increased DNA fragmentation/PARP cleavage). Furthermore, CoQ0 treatment to SKOV-3 xenografted nude mice reduced tumor incidence and burden. Histopathological analyses confirmed that CoQ0 modulated xenografted tumor progression by apoptosis induction. Our findings emphasize that CoQ0 triggered ROS-mediated apoptosis and cytoprotective autophagy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Coenzimas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteína Beclina-1/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
A growing body of research has focused on modifiable risk factors for prevention and attenuation of cognitive decline in aging. This has led to an unprecedented interest in the relationship between diet and cognitive function. Several preclinical and epidemiologic studies suggest that dietary intervention can be used to improve cognitive function but randomized controlled trials are increasingly failing to replicate these findings. Here, we use a canine model of aging to evaluate the effects of specific components of diet supplementation which contain both antioxidants and a combination of mitochondrial cofactors (lipoic acid [LA] and acetyl-l-carnitine) on a battery of cognitive functions. Our data suggest that supplementation with mitochondrial cofactors, but not LA or antioxidant alone, selectively improve long-term recall in aged canines. Furthermore, we found evidence that LA alone could have cognitive impairing effects. These results contrast to those of a previous longitudinal study in aged canine. Our data demonstrate that one reason for this difference may be the nutritional status of animals at baseline for the 2 studies. Overall, this study suggests that social, cognitive, and physical activity together with optimal dietary intake (rather than diet alone) promotes successful brain aging.
Assuntos
Acetilcarnitina/administração & dosagem , Envelhecimento/psicologia , Antioxidantes/administração & dosagem , Coenzimas/administração & dosagem , Transtornos Cognitivos/dietoterapia , Transtornos Cognitivos/prevenção & controle , Cognição/efeitos dos fármacos , Cognição/fisiologia , Suplementos Nutricionais , Ácido Tióctico/administração & dosagem , Acetilcarnitina/farmacologia , Fenômenos Fisiológicos da Nutrição Animal/fisiologia , Animais , Antioxidantes/farmacologia , Coenzimas/farmacologia , Transtornos Cognitivos/psicologia , Modelos Animais de Doenças , Cães , Feminino , Masculino , Memória de Longo Prazo/efeitos dos fármacos , Ácido Tióctico/farmacologiaRESUMO
Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.
Assuntos
Biocatálise/efeitos dos fármacos , Coenzimas/síntese química , Coenzimas/farmacologia , Enzimas/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Coenzimas/química , Coenzimas/metabolismo , Enzimas/química , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Our objectives were to examine if adding metal ion cofactors (COF) to exogenous fibrolytic enzymes (EFE) would increase the beneficial effects of the EFE on the preingestive hydrolysis and in vitro digestibility and fermentation of bermudagrass haylage. In experiment 1, 5 COF (Mn(2+), Co(2+), Fe(2+), Ca(2+), and Mg(2+)) were screened to select the best candidates for synergistically enhancing release of water-soluble carbohydrates (WSC) from bermudagrass haylage by 5 EFE. The 5 EFE (1A, 2A, 11C, 13D, and 15D) were sourced from Trichoderma reesei and Aspergillus oryzae and they were the most effective of 12 EFE at increasing the neutral detergent fiber digestibility of bermudagrass haylage in a previous trial. Adding 1mM of each of the COF to EFE 2A or 11C synergistically increased release of WSC from bermudagrass haylage, as did adding (1mM) Fe(2+) to 1A, Mn(2+), Co(2+), or Fe(2+) to 13D, or Co(2+)or Fe(2+) to 15D. The greatest release of WSC responses were obtained by adding Mn(2+) to 11C (38%) or by adding Fe(2+) to 2A or 13D (10 and 21.9%, respectively). In experiment 2, the effect of increasing the COF dose on in vitro digestibility and fermentation of bermudagrass haylage was examined using the best EFE-COF combinations from experiment 1. Effects of adding increasing doses of these COF on EFE-mediated changes in vitro digestibility depended on the COF-EFE combination. Adding 10mM Mn(2+) alone to bermudagrass haylage increased DMD and NDFD by 2.7 and 6.3% and adding 11C alone increased these measures by 6.6 and 15.5%, respectively. However, adding 10mM Mn(2+) with 11C resulted in 3.5 and 8.1% increases in DMD and NDFD, respectively, beyond the increases caused by adding 11C alone. Adding Fe(2+) to 2A had no effects on EFE-mediated digestibility responses, but 2A prevented adverse effects of adding Fe(2+) alone on DMD and NDFD. In contrast, adding Fe(2+) to 13D reduced the increases in DMD and NDFD caused by adding the EFE alone. This study shows that adding COF to EFE can synergistically increase, decrease, or not affect the hydrolytic effects of EFE on bermudagrass haylage cell walls. The outcome depends on the specific EFE-COF combination and the COF dose. More research is required to understand the mechanisms resulting in these outcomes to exploit beneficial effects of COF on EFE.
Assuntos
Coenzimas/farmacologia , Digestão , Fermentação , Proteínas Fúngicas/metabolismo , Silagem/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Aspergillus oryzae/química , Bovinos , Coenzimas/química , Cynodon/química , Proteínas Fúngicas/química , Hidrólise , Íons/química , Metais/química , Trichoderma/químicaRESUMO
Mitochondria are essential organelles and consequently proper expression and maintenance of the mitochondrial genome are indispensable for proper cell function. The mitochondrial Suv3 (SUPV3L1) helicase is known to have a central role in mitochondrial RNA metabolism and to be essential for maintenance of mitochondrial DNA stability. Here we have performed biochemical investigations to determine the potential regulation of the human Suv3 (hSuv3) helicase function by inorganic cofactors. We find that hSuv3 helicase and ATPase activity in vitro is strictly dependent on the presence of specific divalent cations. Interestingly, we show that divalent cations and nucleotide concentration have a direct effect on helicase substrate stability. Also, hSuv3 helicase is able to utilize several different nucleotide cofactors including both NTPs and dNTPs. Intriguingly, the potency of the individual nucleotide as energy source for hSuv3 unwinding differed depending on the included divalent cation and nucleotide concentration. At low concentrations, all four NTPs could support helicase activity with varying effectiveness depending on the included divalent cation. However, at higher nucleotide concentrations, only ATP was able to elicit the helicase activity of hSuv3. Consequently, we speculate that the capacity of hSuv3 DNA unwinding activity might be sensitive to the local availability of specific inorganic cofactors.
Assuntos
Cátions Bivalentes/farmacologia , Coenzimas/farmacologia , RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Nucleotídeos/farmacologia , Adenosina Trifosfatases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Mitocôndrias/enzimologia , Ligação Proteica/efeitos dos fármacos , Especificidade por SubstratoRESUMO
OBJECTIVE: Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation. BACKGROUND: Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined. METHODS: Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days. RESULTS: Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin. CONCLUSION: We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.
Assuntos
Sobrevivência de Enxerto/fisiologia , Óxido Nítrico Sintase Tipo I/genética , Transplante de Pâncreas , Pâncreas/enzimologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Coenzimas/farmacologia , Isquemia Fria , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/deficiência , Óxido Nítrico Sintase Tipo II/deficiência , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Pâncreas/irrigação sanguínea , Pâncreas/efeitos dos fármacos , Pâncreas/cirurgiaRESUMO
Tyrosine sulfurylation is a post-translational modification important for protein-protein interactions in the extracellular space that are instrumental in cell adhesion, cell signaling, immune responses, and pathogen recognition of host cells. Tyrosine sulfurylation is catalyzed by the tyrosylprotein sulfotransferases (TPSTs), and in humans there are two isoforms: hTPST1 and hTPST2. The study of hTPST function and the development of small molecule probes to examine the role of hTPSTs in cell biology have been delayed by the absence of a continuous direct assay for hTPST activity. We have developed a fluorescent peptide-based assay to directly monitor tyrosine sulfurylation in real time. TPST-mediated tyrosine sulfurylation of the peptides disrupts fluorophore quenching and results in increased fluorescence emission. The assay can be used to study TPST enzymatic activity, and we show that recombinant hTPSTs are active in the absence of divalent metal ions and that optimal activity is at pH 6.0. We further show that the assay can also be used to identify inhibitors of tyrosine sulfurylation. A clear understanding of hTPST function in normal cell biology and in disease states will require the identification of small molecule inhibitors or probes to modulate enzymatic activity, and our results will facilitate that process.
Assuntos
Técnicas Biossensoriais/métodos , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/metabolismo , Peptídeos/metabolismo , Sulfotransferases/metabolismo , Sequência de Aminoácidos , Coenzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/biossínteseRESUMO
Leishmaniasis is one of the major health problems existing globally. The current chemotherapy for leishmaniasis presents several drawbacks like toxicity and increased resistance to existing drugs, and hence, there is a necessity to look out for the novel drug targets and new chemical entities. Current trend in drug discovery arena is the "repurposing" of old drugs for the treatment of diseases. In the present study, an antidepressant, ketanserin, was found lethal to both Leishmania donovani promastigotes and intracellular amastigotes with no apparent toxicity to the cells. Ketanserin killed promastigotes and amastigotes with an IC50 value of 37 µM and 28 µM respectively, in a dose-dependent manner. Ketanserin was found to inhibit L. donovani recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme with an IC50 value of 43 µM. Ketanserin treated promastigotes were exogenously supplemented with sterols like ergosterol and cholesterol to rescue cell death. Ergosterol could recover the inhibition partially, whereas cholesterol supplementation completely failed to rescue the inhibited parasites. Further, HMGR-overexpressing parasites were generated by transfecting Leishmania promastigotes with an episomal pspα hygroα-HMGR construct. Wild-type and HMGR overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. The HMGR overexpressors showed twofold resistance to ketanserin. These observations suggest that the lethal effect of ketanserin is due to inhibition of HMGR, the rate-limiting enzyme of the ergosterol biosynthetic pathway. Since targeting of the sterol biosynthetic pathway enzymes may be useful therapeutically, the present study may have implications in treatment of leishmaniasis.
Assuntos
Antidepressivos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Ketanserina/farmacologia , Leishmania donovani/efeitos dos fármacos , Coenzimas/farmacologiaRESUMO
Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H2-producing catalysts.
Assuntos
Coenzimas/farmacologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Apoenzimas/agonistas , Apoenzimas/química , Apoenzimas/metabolismo , Biocatálise/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Coenzimas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hidrogênio/química , Ferro/química , Proteínas Ferro-Enxofre/agonistas , Modelos MolecularesRESUMO
In vivo, hydrogenases require maturases for active site incorporation. However, in vitro, an active site model with limited catalytic activity could be incorporated into the apo form of [FeFe]-hydrogenase without the aid of maturases, generating enzyme with native activity.
Assuntos
Coenzimas/farmacologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismoRESUMO
Pathologically high brain levels of reactive dicarbonyls such as methylglyoxal or glyoxal initiate processes that lead ultimately to neurodegeneration, presented clinically as Alzheimer's disease and other cognitive or motor impairment disorders. Methylglyoxal and glyoxal result from glycolysis and normal metabolic pathways. Their reaction products with proteins (advanced glycation end products), and their primary chemical toxicities are both linked unequivocally to the primary pathologies of Alzheimer's disease, namely, amyloid plaques and neurofibrillary tangles. Generation of dicarbonyls is countered through the reduction of dicarbonyls by the glutathione-dependent glyoxalase enzyme system. Although glyoxalase-I is overexpressed in early and middle stages of Alzheimer's disease, glutathione depletion in the Alzheimer's afflicted brain cripples its efficacy. Due to the lack of a suitable pharmacological tool, the restoration of glyoxalase enzyme activity in pre-Alzheimer's or manifest Alzheimer's remains yet unvalidated as a means for anti-Alzheimer's therapy development. Disclosed herein are the results of a preclinical study into the therapeutic efficacy of ψ-GSH, a synthetic cofactor of glyoxalase, in mitigating Alzheimer's indicators in a transgenic mouse model (APP/PS1) that is predisposed to Alzheimer's disease. ψ-GSH administration completely averts the development of spatial mnemonic and long-term cognitive/cued-recall impairment. Amyloid ß deposition and oxidative stress indicators are drastically reduced in the ψ-GSH-treated APP/PS1 mouse. ψ-GSH lacks discernible toxicity at strikingly high doses of 2000 mg/kg. The hypothesis that restoring brain glyoxalase activity would ameliorate neurogeneration stands validated, thus presenting a much needed new target for design of anti-Alzheimer's therapeutics. Consequently, ψ-GSH is established as a candidate for drug-development.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Glutationa/análogos & derivados , Lactoilglutationa Liase/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Placa Amiloide/metabolismo , Ureia/análogos & derivados , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Coenzimas/farmacologia , Modelos Animais de Doenças , Glutationa/farmacologia , Produtos Finais de Glicação Avançada/efeitos dos fármacos , Produtos Finais de Glicação Avançada/metabolismo , Glioxal/metabolismo , Lactoilglutationa Liase/fisiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Aldeído Pirúvico/metabolismo , Relação Estrutura-Atividade , Ureia/farmacologiaRESUMO
An L-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with L-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with L-lysine as ligand. The enzyme oxidized L-lysine, L-ornithine and L-arginine, but not other L-amino acids and d-amino acids. The oxidase activity for L-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for L-ornithine and L-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5 kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of L-amino acid oxidases.
Assuntos
L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/metabolismo , Pseudomonas/enzimologia , Sequência de Aminoácidos , Arginina/metabolismo , Arginina/farmacologia , Coenzimas/metabolismo , Coenzimas/farmacologia , Indução Enzimática/efeitos dos fármacos , Flavoproteínas/biossíntese , Flavoproteínas/química , Flavoproteínas/isolamento & purificação , Flavoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , L-Aminoácido Oxidase/biossíntese , L-Aminoácido Oxidase/química , Lisina/metabolismo , Lisina/farmacologia , Dados de Sequência Molecular , Peso Molecular , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Ornitina/metabolismo , Ornitina/farmacologia , Pseudomonas/classificação , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/isolamento & purificação , Especificidade por Substrato , Temperatura , Fatores de TempoRESUMO
The work deals with isolation of transketolase from the rat liver by means of ion-exchange chromatography and substrate elution of enzyme. Experimental data on the regulation of transketolase activity with thiamin pyrophosphate (TPP) and its anticoenzyme analogues are presented. The kinetics of dissociation of holo-TK at pH 4.0 and 5.0 and reactivation of apo-TK at a wide variation of the concentration of TPP and its derivatives with anticoenzyme properties has been studied. The dissociation of holo-TK into apoenzymes and coenzymes at the specified values of pH is characterised by most evident diphasic nature, both fast and slow process being observed. The most part of enzymic activity slowdown falls on the fast phase, while the remaining 20-30% take place within the slow phase. The kinetics research findings illustrate the nonidentity of enzyme active sites with respect to TPP binding with transketolase. The K(m) values for TPP both per the first and second active sites equalled 0.3-4.5 microM and 1.3-19.7 microM, accordingly.
Assuntos
Antimetabólitos/metabolismo , Coenzimas/metabolismo , Holoenzimas/metabolismo , Fígado/enzimologia , Tiamina Pirofosfato/análogos & derivados , Tiamina Pirofosfato/metabolismo , Transcetolase/metabolismo , Animais , Animais não Endogâmicos , Antimetabólitos/farmacologia , Domínio Catalítico , Coenzimas/farmacologia , Ativação Enzimática , Holoenzimas/química , Holoenzimas/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Masculino , Ratos , Tiamina Pirofosfato/farmacologia , Transcetolase/química , Transcetolase/isolamento & purificaçãoRESUMO
Endogenous reactive intermediates such as photoexcited states of tissue chromophores, reactive oxygen species (ROS), reactive carbonyl species (RCS), and transition metal ions are mediators of tissue damage involved in initiation and progression of human pathologies including tumorigenesis, atherosclerosis, diabetes, and neurodegenerative disease. A large body of evidence now suggests that B6 vitamers antagonize the harmful activity of endogenous reactive intermediates fulfilling a very different role than that established as a cofactor for numerous enzymes. In this chapter, the structural basis of vitamin B6 activity as a potent antioxidant, metal chelator, carbonyl scavenger, and photosensitizer is presented and the physiological relevance is discussed.
Assuntos
Coenzimas/fisiologia , Vitamina B 6/fisiologia , Animais , Antioxidantes/farmacologia , Antioxidantes/fisiologia , Quelantes/farmacologia , Coenzimas/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Fármacos Fotossensibilizantes/farmacologia , Piridoxamina/farmacologia , Pele/metabolismo , Pele/efeitos da radiação , Vitamina B 6/farmacologiaRESUMO
vSET (a viral SET domain protein) is an attractive polycomb repressive complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription, as both catalyze histone H3K27 trimethylation. To control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-l-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis, and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a "bump-and-hole" strategy.