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1.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544865

RESUMO

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.


Assuntos
Aminoidrolases/genética , COVID-19/genética , Formiato-Tetra-Hidrofolato Ligase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Complexos Multienzimáticos/genética , Pandemias , Aminoidrolases/antagonistas & inibidores , Animais , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Linhagem Celular , Quirópteros/genética , Quirópteros/virologia , Formiato-Tetra-Hidrofolato Ligase/antagonistas & inibidores , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Antígenos de Histocompatibilidade Menor , Complexos Multienzimáticos/antagonistas & inibidores , Vírus de RNA/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genética
2.
Tohoku J Exp Med ; 255(1): 49-55, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34526430

RESUMO

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.


Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/enzimologia , Eritrócitos/enzimologia , Adolescente , Aminoidrolases/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Regulação para Baixo , Glucosefosfato Desidrogenase/sangue , Glutationa/sangue , Glicólise , Humanos , Lactente , Japão , Via de Pentose Fosfato , Regulação para Cima
3.
J Med Chem ; 64(15): 11288-11301, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34337952

RESUMO

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays an important role in one-carbon metabolism. The MTHFD2 gene is upregulated in various cancers but very low or undetectable in normal proliferating cells, and therefore a potential target for cancer treatment. In this study, we present the structure of MTHFD2 in complex with xanthine derivative 15, which allosterically binds to MTHFD2 and coexists with the substrate analogue. A kinetic study demonstrated the uncompetitive inhibition of MTHFD2 by 15. Allosteric inhibitors often provide good selectivity and, indeed, xanthine derivatives are highly selective for MTHFD2. Moreover, several conformational changes were observed upon the binding of 15, which impeded the binding of the cofactor and phosphate to MTHFD2. To the best of our knowledge, this is the first study to identify allosteric inhibitors targeting the MTHFD family and our results would provide insights on the inhibition mechanism of MTHFD proteins and the development of novel inhibitors.


Assuntos
Aminoidrolases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Enzimas Multifuncionais/antagonistas & inibidores , Xantina/farmacologia , Sítio Alostérico/efeitos dos fármacos , Aminoidrolases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Modelos Moleculares , Estrutura Molecular , Enzimas Multifuncionais/metabolismo , Relação Estrutura-Atividade , Xantina/síntese química , Xantina/química
4.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34244426

RESUMO

Cancer cells acquire metabolic reprogramming to satisfy their high biogenetic demands, but little is known about how metabolic remodeling enables cancer cells to survive stress associated with genomic instability. Here, we show that the mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) is transcriptionally suppressed by p53, and its up-regulation by p53 inactivation leads to increased folate metabolism, de novo purine synthesis, and tumor growth in vivo and in vitro. Moreover, MTHFD2 unexpectedly promotes nonhomologous end joining in response to DNA damage by forming a complex with PARP3 to enhance its ribosylation, and the introduction of a PARP3-binding but enzymatically inactive MTHFD2 mutant (e.g., D155A) sufficiently prevents DNA damage. Notably, MTHFD2 depletion strongly restrains p53-deficient cell proliferation and sensitizes cells to chemotherapeutic agents, indicating a potential role for MTHFD2 depletion in the treatment of p53-deficient tumors.


Assuntos
Aminoidrolases/genética , Dano ao DNA , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Transcrição Genética , Proteína Supressora de Tumor p53/deficiência , Adenilato Quinase/metabolismo , Aminoidrolases/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Carbono/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Enzimas Multifuncionais/metabolismo , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
5.
Commun Biol ; 4(1): 882, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272468

RESUMO

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.


Assuntos
Aminoidrolases/genética , Citosina , Expressão Gênica , Processamento de Proteína , Aminoidrolases/metabolismo , Citosina/metabolismo , Edição de Genes , Humanos
6.
Biotechnol J ; 16(10): e2100010, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34270173

RESUMO

In recent years, many biocatalytic processes have been developed for the production of chemicals and pharmaceuticals. In this context, enzyme immobilization methods have attracted attention for their advantages, such as continuous production and increased stability. Here, enzyme immobilization methods and a collection of nitrilases from biodiversity for the conversion of 3-cyanopyridine to nicotinic acid were screened. Substrate conversion over 10 conversion cycles was monitored to optimize the process. The best immobilization conditions were found with cross-linking using glutaraldehyde to modify the PMMA beads. This method showed good activity over 10 cycles in a batch reactor at 30 and 40°C. Finally, production with a new thermostable nitrilase was examined in a continuous packed bed reactor, showing very high stability of the biocatalytic process at a flow rate of 0.12 ml min-1 and a temperature of 50°C. The complete conversion of 3-cyanopyridine was obtained over 30 days of operation. Future steps will concern reactor scale-up to increase the production rate with reasonable pressure drops.


Assuntos
Niacina , Aminoidrolases/metabolismo , Biocatálise , Enzimas Imobilizadas
7.
Nat Commun ; 12(1): 2748, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980881

RESUMO

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.


Assuntos
Regulação Alostérica , Aminoidrolases/química , Aminoidrolases/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glutamina/metabolismo , Hidrólise , Imidazóis/metabolismo , Espectroscopia de Ressonância Magnética , Complexos Multienzimáticos , Mutação , Conformação Proteica , Ribonucleotídeos/metabolismo , Thermotoga maritima/enzimologia
8.
Sci Rep ; 11(1): 9647, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958623

RESUMO

The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme-HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.


Assuntos
Aminoidrolases/metabolismo , Histidina/biossíntese , Pirofosfatases/metabolismo , Monofosfato de Adenosina/metabolismo , Aminoidrolases/química , Aminoidrolases/genética , Catálise , Domínio Catalítico , Medicago truncatula/enzimologia , Medicago truncatula/metabolismo , Redes e Vias Metabólicas , Filogenia , Estrutura Terciária de Proteína , Pirofosfatases/química , Pirofosfatases/genética , Alinhamento de Sequência
9.
J Biol Chem ; 296: 100651, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839153

RESUMO

The Rid protein family (PF14588, IPR006175) is divided into nine subfamilies, of which only the RidA subfamily has been characterized biochemically. RutC, the founding member of one subfamily, is encoded in the pyrimidine utilization (rut) operon that encodes a pathway that allows Escherichia coli to use uracil as a sole nitrogen source. Results reported herein demonstrate that RutC has 3-aminoacrylate deaminase activity and facilitates one of the reactions previously presumed to occur spontaneously in vivo. RutC was active with several enamine-imine substrates, showing similarities and differences in substrate specificity with the canonical member of the Rid superfamily, Salmonella enterica RidA. Under standard laboratory conditions, a Rut pathway lacking RutC generates sufficient nitrogen from uracil for growth of E. coli. These results support a revised model of the Rut pathway and provide evidence that Rid proteins may modulate metabolic fitness, rather than catalyzing essential functions.


Assuntos
Acrilatos/metabolismo , Aminoidrolases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases/metabolismo , Aminoidrolases/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Família , Nitrogênio/metabolismo , Oxirredutases/genética , Fosfato de Piridoxal/metabolismo , Salmonella enterica/enzimologia , Especificidade por Substrato , Uracila/metabolismo
10.
Anal Bioanal Chem ; 413(13): 3573-3582, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33829277

RESUMO

Mammalian folate-dependent one-carbon (1C) metabolism provides the building blocks essential during development via amino acid interconversion, methyl-donor production, regeneration of redox factors, and de novo purine and thymidylate synthesis. Folate supplementation prevents many neural tube defects (NTDs) that occur during the embryonic process of neurulation. The mechanism by which folate functions during neurulation is not well understood, and not all NTDs are preventable by folate supplementation. Mthfd1l is a mitochondrial 1C metabolism enzyme that produces formate, a 1C donor that fuels biosynthesis and the methyl cycle in the cytoplasm. Homozygous deletion of the Mthfd1l gene in mice (Mthfd1lz/z) causes embryonic lethality, developmental delay, and folate-resistant NTDs. These mice also have defects in cranial mesenchyme formation. In this work, mass spectrometry imaging was used to obtain ion maps of the cranial mesenchyme that identified the spatial distribution and relative abundance of metabolites in wild-type and Mthfd1lz/z embryos. The relative abundances of purine and thymidylate derivatives, as well as amino acids, were diminished in the cranial mesenchyme of Mthfd1lz/z embryos. Loss of Mthfd1l activity in this region also led to abnormal levels of methionine and dysregulated energy metabolism. These alterations in metabolism suggest possible approaches to preventing NTDs in humans.


Assuntos
Aminoidrolases/genética , Formiato-Tetra-Hidrofolato Ligase/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Complexos Multienzimáticos/genética , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Camundongos , Camundongos Knockout
11.
Sci Rep ; 11(1): 9107, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907262

RESUMO

Theileria equi, an intraerythrocytic protozoan parasite, causes equine piroplasmosis, a disease which negatively impacts the global horse industry. Genetic manipulation is one of the research tools under development as a control method for protozoan parasites, but this technique needs to be established for T. equi. Herein, we report on the first development of a stable transgenic T. equi line expressing enhanced green fluorescent protein/blasticidin S deaminase (eGFP/BSD). To express the exogenous fusion gene in T. equi, regulatory regions of the elongation factor-1 alpha (ef-1α) gene were identified in T. equi. An eGFP/BSD-expression cassette containing the ef-1α gene promoter and terminator regions was constructed and integrated into the T. equi genome. On day 9 post-transfection, blasticidin-resistant T. equi emerged. In the clonal line of T. equi obtained by limiting dilution, integration of the eGFP/BSD-expression cassette was confirmed in the designated B-locus of the ef-1α gene via PCR and Southern blot analyses. Parasitaemia dynamics between the transgenic and parental T. equi lines were comparable in vitro. The eGFP/BSD-expressing transgenic T. equi and the methodology used to generate it offer new opportunities for better understanding of T. equi biology, with the add-on possibility of discovering effective control methods against equine piroplasmosis.


Assuntos
Aminoidrolases/genética , Proteínas de Fluorescência Verde/genética , Organismos Geneticamente Modificados , Theileria/genética , Regulação da Expressão Gênica , Nucleosídeos/farmacologia , Fator 1 de Elongação de Peptídeos/genética , Plasmídeos , Theileria/efeitos dos fármacos , Transfecção
12.
Nat Commun ; 12(1): 1940, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782411

RESUMO

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.


Assuntos
Aminoidrolases/genética , Antígeno B7-H1/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Neoplasias Pancreáticas/genética , Processamento de Proteína Pós-Traducional , Linfócitos T Citotóxicos/imunologia , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Carcinogênese/imunologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Embrião de Mamíferos , Fibroblastos/imunologia , Fibroblastos/patologia , Ácido Fólico/imunologia , Ácido Fólico/metabolismo , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Linfócitos T Citotóxicos/patologia , Carga Tumoral , Evasão Tumoral , Uridina Difosfato N-Acetilglicosamina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Theranostics ; 11(8): 3661-3675, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33664854

RESUMO

Rationale: Lung adenocarcinoma (LUAD) is an aggressive disease with high propensity of metastasis. Among patients with early-stage disease, more than 30% of them may relapse or develop metastasis. There is an unmet medical need to stratify patients with early-stage LUAD according to their risk of relapse/metastasis to guide preventive or therapeutic approaches. In this study, we identified 4 genes that can serve both therapeutic and diagnostic (theranostic) purposes. Methods: Three independent datasets (GEO, TCGA, and KMPlotter) were used to evaluate gene expression profile of patients with LUAD by unbiased screening approach. Upon significant genes uncovered, functional enrichment analysis was carried out. The predictive power of their expression on patient prognosis were evaluated. Once confirmed their theranostic roles by integrated bioinformatics, we further conducted in vitro and in vivo validation. Results: We found that four genes (ADAM9, MTHFD2, RRM2, and SLC2A1) were associated with poor patient outcomes with an increased hazard ratio in LUAD. Knockdown of them, both separately and simultaneously, suppressed lung cancer cell proliferation and migration ability in vitro and prolonged survival time in metastatic tumor mouse models. Moreover, these four biomarkers were found to be overexpressed in tumor tissues from LUAD patients, and the total immunohistochemical staining scores correlated with poor prognosis. Conclusions: These results suggest that these four identified genes could be theranostic biomarkers for stratifying high-risk patients who develop relapse/metastasis in early-stage LUAD. Developing therapeutic approaches for the four biomarkers may benefit early-stage LUAD patients after surgery.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/secundário , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Células A549 , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Adenocarcinoma de Pulmão/cirurgia , Aminoidrolases/antagonistas & inibidores , Aminoidrolases/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/cirurgia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/antagonistas & inibidores , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos SCID , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/genética , Medicina de Precisão , Prognóstico , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Fatores de Risco , Transcriptoma , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Sci Rep ; 11(1): 6606, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758248

RESUMO

High lead (Pb) concentration in soils is becoming a severe threat to human health. It also deteriorates plants, growth, yield and quality of food. Although the use of plant growth-promoting rhizobacteria (PGPR), biochar and compost can be effective environment-friendly amendments for decreasing Pb stress in crop plants, the impacts of their simultaneous co-application has not been well documented. Thus current study was carried, was conducted to investigate the role of rhizobacteria and compost mixed biochar (CB) under Pb stress on selected soil properties and agronomic parameters in mint (Mentha piperita L.) plants. To this end, six treatments were studied: Alcaligenes faecalis, Bacillus amyloliquefaciens, CB, PGPR1 + CB, PGPR2 + CB and control. Results showed that the application A. faecalis + CB significantly decreased soil pH and EC over control. However, OM, nitrogen, phosphorus and potassium concentration were significantly improved in the soil where A. faecalis + CB was applied over control. The A. faecalis + CB treatment significantly improved mint plant root dry weight (58%), leaves dry weight (32%), chlorophyll (37%), and N (46%), P (39%) and K (63%) leave concentration, while also decreasing the leaves Pb uptake by 13.5% when compared to the unamended control. In conclusion, A. faecalis + CB has a greater potential to improve overall soil quality, fertility and mint plant productivity under high Pb soil concentration compared to the sole application of CB and A. faecalis.


Assuntos
Carvão Vegetal/metabolismo , Compostagem/métodos , Chumbo/toxicidade , Mentha/efeitos dos fármacos , Rizosfera , Poluentes do Solo/toxicidade , Alcaligenes faecalis/enzimologia , Alcaligenes faecalis/metabolismo , Aminoidrolases/metabolismo , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Frutas/química , Chumbo/metabolismo , Mentha/microbiologia , Poluentes do Solo/metabolismo , Estresse Fisiológico , Verduras/química
15.
Biomed Pharmacother ; 137: 111309, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33524784

RESUMO

Anti-Koch and HAART have been shown to independently induce toxicity to the liver and kidney, albeit available data are few and inconsistent. The present study evaluates the impact of Anti-Koch and HAART, when administered singly and in combination, on hepatic and renal status, and the possible role of adenine deaminase (ADA)/xanthine oxidase (XO) pathway. Anti-Koch and HAART administration were observed to independently impair hepatic and renal functions, diminish glutathione content, and substantially increase lipid peroxidation (MDA) and nitrogen reactive specie (NO). Coherently, these drugs caused significant accumulation of polymorphonuclear leucocytes, up-regulated ADA/XO signaling, increased uric acid production, and enhanced DNA fragmentation in the liver and kidney. Anti-Koch treatment did not significantly alter hepatic and renal levels of nitric oxide nor induce DNA fragmentation in the kidney. Co-administration of anti-Koch and HAART aggravated the observed biochemical alterations. Findings from the histopathological studies of the liver and renal tissues were in agreement with observed biochemical alterations. In conclusion, this report is the first to reveal that anti-Koch and HAART, when administered singly or in combination, attenuate glutathione content and elevate uric acid production in the liver and kidney via upregulation of ADA/XO signaling with resultant oxidative and nitrosative stress, and increased DNA fragmentation.


Assuntos
Terapia Antirretroviral de Alta Atividade/efeitos adversos , Antituberculosos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutationa/metabolismo , Nefropatias/induzido quimicamente , Nefropatias/patologia , Ácido Úrico/metabolismo , Aminoidrolases/metabolismo , Animais , Fragmentação do DNA , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Nitrogênio/metabolismo , Xantina Oxidase/metabolismo
16.
Acta Neuropathol Commun ; 9(1): 16, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33468252

RESUMO

Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from nutrient starvation. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in GBM cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around "pseudopalisading necrosis." Genetic suppression of MTHFD2 and autophagy inhibition caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation. These results highlight a critical role for serine-dependent one-carbon metabolism in surviving glutamine starvation and suggest new therapeutic targets for glioma cells adapting to a low-nutrient microenvironment.


Assuntos
Aminoidrolases/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glutamina/metabolismo , Glicina/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Enzimas Multifuncionais/genética , Serina/metabolismo , Aminoidrolases/metabolismo , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Metabolômica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Enzimas Multifuncionais/metabolismo , Microambiente Tumoral/genética , Regulação para Cima
17.
Microb Cell Fact ; 20(1): 11, 2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33422055

RESUMO

BACKGROUND: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli. RESULTS: To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM L-Phe to D-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic L-amino acids to their corresponding D-amino acids. CONCLUSIONS: The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of D-amino acids via stereoinversion.


Assuntos
Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Aminoidrolases/metabolismo , Formiato Desidrogenases/metabolismo , Biocatálise , Burkholderia/enzimologia , Clostridiales/enzimologia , Proteus mirabilis/enzimologia , Estereoisomerismo , Especificidade por Substrato
18.
Chemphyschem ; 22(8): 796-804, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33463886

RESUMO

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α-ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α-ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacyl-enzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental kcat (17.67 kcal/mol).


Assuntos
Aminoidrolases/metabolismo , Cisteína/metabolismo , Teoria da Densidade Funcional , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Aminoidrolases/química , Animais , Biocatálise , Cisteína/química , Ácido Glutâmico/química , Lisina/química , Camundongos , Modelos Moleculares
19.
Proteins ; 89(3): 336-347, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33118210

RESUMO

Predicting the range of substrates accepted by an enzyme from its amino acid sequence is challenging. Although sequence- and structure-based annotation approaches are often accurate for predicting broad categories of substrate specificity, they generally cannot predict which specific molecules will be accepted as substrates for a given enzyme, particularly within a class of closely related molecules. Combining targeted experimental activity data with structural modeling, ligand docking, and physicochemical properties of proteins and ligands with various machine learning models provides complementary information that can lead to accurate predictions of substrate scope for related enzymes. Here we describe such an approach that can predict the substrate scope of bacterial nitrilases, which catalyze the hydrolysis of nitrile compounds to the corresponding carboxylic acids and ammonia. Each of the four machine learning models (logistic regression, random forest, gradient-boosted decision trees, and support vector machines) performed similarly (average ROC = 0.9, average accuracy = ~82%) for predicting substrate scope for this dataset, although random forest offers some advantages. This approach is intended to be highly modular with respect to physicochemical property calculations and software used for structural modeling and docking.


Assuntos
Aminoidrolases , Proteínas de Bactérias , Aprendizado de Máquina , Simulação de Acoplamento Molecular/métodos , Aminoidrolases/química , Aminoidrolases/genética , Aminoidrolases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Fenômenos Químicos , Ligantes , Nitrilas/química , Nitrilas/metabolismo , Ligação Proteica
20.
Angew Chem Int Ed Engl ; 60(7): 3679-3684, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33141478

RESUMO

A mirror-image strategy, that is, symmetry analysis of the substrate-binding pocket, was applied to identify two key amino acid residues W170 and V198 that possibly modulate the enantiopreference of a nitrilase from Synechocystis sp. PCC6803 towards 3-isobutyl glutaronitrile (1 a). Exchange of these two residues resulted in the enantiopreference inversion (S, 90 % ee to R, 47 % ee). By further reshaping the substrate-binding pocket via routine site-saturation and combinatorial mutagenesis, variant E8 with higher activity and stereoselectivity (99 % ee, R) was obtained. The mutant enzyme was applied in the preparation of optically pure (R)-3-isobutyl-4-cyanobutanoic acid ((R)-2 a) and showed similar stereopreference inversion towards a series of 3-substituted glutaronitriles. This study may offer a general strategy to switch the stereopreference of other nitrilases and other enzymes toward the desymmetric reactions of prochiral substrates with two identical reactive functional groups.


Assuntos
Aminoidrolases/metabolismo , Nitrilas/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Biocatálise , Hidrólise , Estrutura Molecular , Nitrilas/química , Estereoisomerismo , Synechocystis/enzimologia
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