Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.336
Filtrar
2.
Enzyme Microb Technol ; 133: 109446, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31874692

RESUMO

Glutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced l-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5 mM glutaric acid was produced from 800 mM 5-aminovaleric acid using only 120 mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.


Assuntos
Aminoácido Oxirredutases/metabolismo , Escherichia coli/enzimologia , Glutaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Catalase/metabolismo , Escherichia coli/genética , Fermentação , Engenharia Metabólica/métodos
3.
Mol Cell ; 76(4): 660-675.e9, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31542297

RESUMO

Histone posttranslational modifications (PTMs) regulate chromatin structure and dynamics during various DNA-associated processes. Here, we report that lysine glutarylation (Kglu) occurs at 27 lysine residues on human core histones. Using semi-synthetic glutarylated histones, we show that an evolutionarily conserved Kglu at histone H4K91 destabilizes nucleosome in vitro. In Saccharomyces cerevisiae, the replacement of H4K91 by glutamate that mimics Kglu influences chromatin structure and thereby results in a global upregulation of transcription and defects in cell-cycle progression, DNA damage repair, and telomere silencing. In mammalian cells, H4K91glu is mainly enriched at promoter regions of highly expressed genes. A downregulation of H4K91glu is tightly associated with chromatin condensation during mitosis and in response to DNA damage. The cellular dynamics of H4K91glu is controlled by Sirt7 as a deglutarylase and KAT2A as a glutaryltransferase. This study designates a new histone mark (Kglu) as a new regulatory mechanism for chromatin dynamics.


Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , Glutaratos/metabolismo , Histonas/metabolismo , Mitose , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Lisina , Camundongos , Nucleossomos/genética , Células RAW 264.7 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Fatores de Tempo
4.
Eur J Pharm Sci ; 140: 105072, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518680

RESUMO

Isocitrate dehydrogenase 1 mutations have been discovered in an array of hematologic malignancies and solid tumors. These mutations could cause the production of high levels of 2-hydroxyglutarate, which in turn implicated in epigenetic changes and impaired cell differentiation. Here, we described the characterization of compound I-8, a novel mutant IDH1 inhibitor, both in vitro and in vivo. Compound I-8 specifically inhibited 2-HG production, reduced histone methylation levels, induced differentiation and depleted stem characteristics in engineered and endogenous IDH1 mutant cells. In addition, oral administration of I-8 also significantly suppressed 2-HG production and histone methylation with dose of 150 mg/kg. And I-8 treatment also could induce differentiation and attenuate stem characteristics in tumor tissue. Together, these studies indicated that compound I-8 has clinical potential in tumor therapies as a effective mutant IDH1 inhibitor, and provided scientific guidance for the development of mutant IDH1 inhibitor in the future.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Isocitrato Desidrogenase/antagonistas & inibidores , Actinas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Epigênese Genética , Glutaratos/metabolismo , Histonas/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Metilação/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Conformação Proteica
5.
mBio ; 10(4)2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363033

RESUMO

Glutarate, a metabolic intermediate in the catabolism of several amino acids and aromatic compounds, can be catabolized through both the glutarate hydroxylation pathway and the glutaryl-coenzyme A (glutaryl-CoA) dehydrogenation pathway in Pseudomonas putida KT2440. The elucidation of the regulatory mechanism could greatly aid in the design of biotechnological alternatives for glutarate production. In this study, it was found that a GntR family protein, CsiR, and a LysR family protein, GcdR, regulate the catabolism of glutarate by repressing the transcription of csiD and lhgO, two key genes in the glutarate hydroxylation pathway, and by activating the transcription of gcdH and gcoT, two key genes in the glutaryl-CoA dehydrogenation pathway, respectively. Our data suggest that CsiR and GcdR are independent and that there is no cross-regulation between the two pathways. l-2-Hydroxyglutarate (l-2-HG), a metabolic intermediate in the glutarate catabolism with various physiological functions, has never been elucidated in terms of its metabolic regulation. Here, we reveal that two molecules, glutarate and l-2-HG, act as effectors of CsiR and that P. putida KT2440 uses CsiR to sense glutarate and l-2-HG and to utilize them effectively. This report broadens our understanding of the bacterial regulatory mechanisms of glutarate and l-2-HG catabolism and may help to identify regulators of l-2-HG catabolism in other species.IMPORTANCE Glutarate is an attractive dicarboxylate with various applications. Clarification of the regulatory mechanism of glutarate catabolism could help to block the glutarate catabolic pathways, thereby improving glutarate production through biotechnological routes. Glutarate is a toxic metabolite in humans, and its accumulation leads to a hereditary metabolic disorder, glutaric aciduria type I. The elucidation of the functions of CsiR and GcdR as regulators that respond to glutarate could help in the design of glutarate biosensors for the rapid detection of glutarate in patients with glutaric aciduria type I. In addition, CsiR was identified as a regulator that also regulates l-2-HG metabolism. The identification of CsiR as a regulator that responds to l-2-HG could help in the discovery and investigation of other regulatory proteins involved in l-2-HG catabolism.


Assuntos
Glutaratos/metabolismo , Pseudomonas putida/metabolismo , Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Transcrição/metabolismo
6.
Oncogene ; 38(42): 6835-6849, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31406254

RESUMO

Chondrosarcoma is the second most common malignant bone tumor. It is characterized by low vascularity and an abundant extracellular matrix, which confer these tumors resistance to chemotherapy and radiotherapy. There are currently no effective treatment options for relapsed or dedifferentiated chondrosarcoma, and new targeted therapies need to be identified. Isocitrate dehydrogenase (IDH) mutations, which are detected in ~50% of chondrosarcoma patients, contribute to malignant transformation by catalyzing the production of 2-hydroxyglutarate (2-HG), a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Mutant IDH inhibitors are therefore potential novel anticancer drugs in IDH mutant tumors. Here, we examined the efficacy of the inhibition of mutant IDH1 as an antitumor approach in chondrosarcoma cells in vitro and in vivo, and investigated the association between the IDH mutation and chondrosarcoma cells. DS-1001b, a novel, orally bioavailable, selective mutant IDH1 inhibitor, impaired the proliferation of chondrosarcoma cells with IDH1 mutations in vitro and in vivo, and decreased 2-HG levels. RNA-seq analysis showed that inhibition of mutant IDH1 promoted chondrocyte differentiation in the conventional chondrosarcoma L835 cell line and caused cell cycle arrest in the dedifferentiated JJ012 cell line. Mutant IDH1-mediated modulation of SOX9 and CDKN1C expression regulated chondrosarcoma tumor progression, and DS-1001b upregulated the expression of these genes via a common mechanism involving the demethylation of H3K9me3. DS-1001b treatment reversed the epigenetic changes caused by aberrant histone modifications. The present data strongly suggest that inhibition of mutant IDH1 is a promising therapeutic approach in chondrosarcoma, particularly for the treatment of relapsed or dedifferentiated chondrosarcoma.


Assuntos
Neoplasias Ósseas/patologia , Condrossarcoma/patologia , Inibidores Enzimáticos/farmacologia , Código das Histonas , Isocitrato Desidrogenase/antagonistas & inibidores , Mutação , Neoplasias Ósseas/metabolismo , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Proliferação de Células , Condrossarcoma/metabolismo , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Fatores de Transcrição SOX9/metabolismo
7.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140255, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31349060

RESUMO

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.


Assuntos
Oxirredutases do Álcool/química , Encefalopatias Metabólicas Congênitas/enzimologia , Mutação de Sentido Incorreto , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Substituição de Aminoácidos , Encefalopatias Metabólicas Congênitas/genética , Catálise , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/metabolismo , Glutaratos/química , Glutaratos/metabolismo , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismo
8.
Nat Commun ; 10(1): 3337, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350399

RESUMO

Various biosynthetic pathways have been designed to explore sustainable production of glutarate, an attractive C5 building block of polyesters and polyamides. However, its efficient production has not been achieved in Escherichia coli. Here, we use E. coli native lysine catabolic machinery for glutarate biosynthesis. This endogenous genes-only design can generate strong metabolic driving force to maximize carbon flux toward glutarate biosynthesis by replenishing glutamate and NAD(P)H for lysine biosynthesis, releasing lysine feedback inhibition, and boosting oxaloacetate supply. We use native transporters to overcome extracellular accumulation of cadaverine and 5-aminovalerate. With these efforts, both high titer (54.5 g L-1) and high yield (0.54 mol mol-1 glucose) of glutarate production are achieved under fed-batch conditions. This work demonstrates the power of redirecting carbon flux and the role of transporters to decrease intermediate accumulation.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Glutaratos/metabolismo , Lisina/metabolismo , Vias Biossintéticas , Ácido Glutâmico/metabolismo , Engenharia Metabólica , NADP/metabolismo
9.
Anal Chim Acta ; 1077: 174-182, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307707

RESUMO

With the rapid development of immunometabolism, 2-hydroxyglutarate (2-HG) is being promoted as a key immunometabolite to regulate the immune system. Based on the well-established crosstalk between 2-HG and other immunometabolites, here we firstly constructed a 2-HG metabolic panel by mapping the related metabolic pathways. Quantitative methods to globally monitor 2-HG metabolic panel are of great importance for immunometabolism study. However, the existence of enantiomer hampers the accurate measurement of these immunometabolites. This study addressed an original isotopically-paired chiral derivatization approach for UPLC-MS/MS quantification of 2-HG metabolic panel. To achieve better chromatographic separation, N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was utilized as an optical resolving reagent to form diastereomers. For accurate quantitation, an 18O2-labeled-TSPC reagent was designed and readily synthesized to produce one-to-one internal standards. The developed approach enabled an accurate quantification of 13 immunometabolites in 2-HG metabolic panel with good linearity (R2 > 0.99) and high sensitivity (0.5-120 fmol for LLOQ). With this method, we were able to simultaneously monitor the specific alterations of 2-HG metabolic panel in collagen-induced rheumatoid arthritis (CIA) rats. The measured levels of this panel ranged from 0.02 to 85.14 µg g-1 for synovium tissue and 0.012 to 87.75 µmol L-1 for serum samples. We envisage that the present isotopically-paired chiral derivatization approach will be practicable for different bio-samples to quantitatively profile the amino- and hydroxyl acids submetabolome, especially for the endogenous enantiomers. By virtue of the low cost of reagents and the simple procedure used in the assay, this method could be readily implemented.


Assuntos
Glutaratos/metabolismo , Fenilalanina/análogos & derivados , Animais , Cromatografia Líquida/métodos , Feminino , Marcação por Isótopo , Limite de Detecção , Metabolômica/métodos , Oxigênio/química , Isótopos de Oxigênio/química , Ratos Wistar , Reprodutibilidade dos Testes , Estereoisomerismo , Espectrometria de Massas em Tandem/métodos
11.
Rapid Commun Mass Spectrom ; 33(17): 1401-1409, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31148247

RESUMO

RATIONALE: 2-Hydroxyglutarate (2-hg) exists as enantiomers and can readily undergo cyclization to its lactone. Gas chromatography/electron ionization mass spectrometry (GC/EI-MS) has been used to separate 2-hg enantiomers in bodily fluids but the assay cannot simultaneously measure cyclic and acylic 2-hg enantiomers. Furthermore, the assignment of ion structures was not verified by complementary MS data. METHODS: GC/EI-MS and product ion analysis were used to obtain MS and MS/MS spectra of 2-hg, deuterated and 13 C-labeled 2-hg, and 2-hg lactone. Ion structures and EI fragmentation mechanisms were determined by fragmentation pattern and isotopologue comparisons. Using the EI data, a GC/MS/MS assay was developed to separate and detect 2-hg enantiomers and 2-hg lactone enantiomers in blood and urine using a cyclodextrin capillary column. RESULTS: A new ion structure was predicted for the 85 m/z fragment than what was previously hypothesized, and the 117 m/z ion was the only fragment unique to the linear 2-hg compound. MS/MS data suggested that the majority of the fragments were the result of secondary fragmentation. Finally, separation of serum and urine 2-hg and 2-hg lactone enantiomers was achieved, and the acyclic 2-hg compound was found to be the major compound detected, though the amount of lactone detected was considerable in a number of samples. CONCLUSIONS: Unique EI fragmentation pathways for both 2-hg and the 2-hg lactone have been described. Subsequently, the GC/MS/MS assay presented herein has significant potential as a novel clinical assay as it separates and detects both 2-hg enantiomers and the 2-hg lactone enantiomers, a capability which has not been previously demonstrated by any other assay to date.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Glutaratos/química , Lactonas/química , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Espectrometria de Massas em Tandem/métodos
12.
Proc Natl Acad Sci U S A ; 116(26): 12851-12856, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31182575

RESUMO

Oncogenic IDH1/2 mutations produce 2-hydroxyglutarate (2HG), resulting in competitive inhibition of DNA and protein demethylation. IDH-mutant cancer cells show an inability to differentiate but whether 2HG accumulation is sufficient to perturb differentiation directed by lineage-specifying transcription factors is unknown. A MyoD-driven model was used to study the role of IDH mutations in the differentiation of mesenchymal cells. The presence of 2HG produced by oncogenic IDH2 blocks the ability of MyoD to drive differentiation into myotubes. DNA 5mC hypermethylation is dispensable while H3K9 hypermethylation is required for this differentiation block. IDH2-R172K mutation results in H3K9 hypermethylation and impaired accessibility at myogenic chromatin regions but does not result in genome-wide decrease in accessibility. The results demonstrate the ability of the oncometabolite 2HG to block transcription factor-mediated differentiation in a molecularly defined system.


Assuntos
Diferenciação Celular , Glutaratos/metabolismo , Histonas/metabolismo , Proteína MyoD/metabolismo , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina , Metilação de DNA , Glutaratos/farmacologia , Isocitrato Desidrogenase/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mutação
13.
Enzyme Microb Technol ; 128: 72-78, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186113

RESUMO

Glutaric acid is an attractive C5 dicarboxylic acid with wide applications in the biochemical industry. Glutaric acid can be produced by fermentation and bioconversion, and several of its biosynthesis pathways have been well characterized, especially the simple pathway involving glutaric acid from l-lysine using 5-aminovaleric acid. We previously reported the production of glutaric acid using 5-aminovaleric acid and α-ketoglutaric acid by a whole-cell reaction, resulting in a high conversion yield. In this study, we sought to enhance the stability and reusability of this whole-cell system for realizing the efficient production of glutaric acid under harsh reaction conditions. To this end, various matrices were screened to immobilize Escherichia coli whole-cell overexpressing 4-aminobutyrate aminotransferase (GabT), succinate semi-aldehyde dehydrogenase (GabD), and NAD(P)H oxidase (NOX). We ultimately selected a PVA-PEG gel (LentiKats®) for cell entrapment, and several factors of the reaction were optimized. The optimal temperature and pH were 35 °C and 8.5, respectively. Treatment with Tween 80 as a surfactant, as well as additional NOX, was found to be effective. Under the optimized conditions, an immobilized cell retained 55% of its initial activity even after the eighth cycle, achieving 995.2 mM accumulated glutaric acid, whereas free cell lost most of their activity after only two cycles. This optimized whole-cell system can be used in the large-scale production of glutaric acid.


Assuntos
Aminoácidos Neutros/metabolismo , Células Imobilizadas/metabolismo , Escherichia coli/metabolismo , Glutaratos/metabolismo , Biotransformação , Escherichia coli/enzimologia , Géis , Concentração de Íons de Hidrogênio , Polietilenoglicóis , Álcool de Polivinil , Temperatura
14.
Methods Mol Biol ; 1978: 155-165, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119662

RESUMO

The fruit fly Drosophila melanogaster has emerged as an ideal system in which to study 2-hydroxyglutarate (2HG) metabolism. Unlike many mammalian tissues and cell lines, which primarily accumulate D- or L-2HG as the result of genetic mutations or metabolic stress, Drosophila larvae accumulate high concentrations of L-2HG during normal larval growth. As a result, flies represent one of the few model systems that allows for studies of endogenous L-2HG metabolism. Moreover, the Drosophila genome not only encodes key enzymes involved in the synthesis and degradation of D-2HG, but the fly has also been used as to investigate the in vivo effects of oncogenic isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations. All of these studies, however, rely on mass spectrometry-based methods to distinguish between the D- and L-2HG enantiomers. While such approaches are common among labs studying mammalian cell culture, few Drosophila studies have attempted to resolve and measure the individual 2HG enantiomers. Here we describe a highly reproducible gas chromatography-mass spectrometry (GC-MS)-based protocol that allows for quantitative measurements of both 2HG enantiomers in Drosophila homogenates.


Assuntos
Drosophila melanogaster/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glutaratos/isolamento & purificação , Animais , Drosophila melanogaster/metabolismo , Glutaratos/química , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Larva/química , Larva/genética , Mutação , Estereoisomerismo
15.
Mol Neurobiol ; 56(11): 7694-7707, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31104295

RESUMO

Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 µm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encefalopatias Metabólicas/patologia , Movimento Celular/efeitos dos fármacos , Glutaratos/farmacologia , Glutaril-CoA Desidrogenase/deficiência , Pericitos/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Capilares/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/patologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
16.
Radiology ; 291(3): 752-762, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30990380

RESUMO

Background Isocitrate dehydrogenase (IDH) mutation has become one of the most important prognostic biomarkers in glioma management. Measurement of 2-hydroxyglutarate (2HG) with MR spectroscopy has shown high pooled sensitivity, although false-positive results with MR spectroscopy have been reported. Purpose To investigate factors associated with false-positive 2HG measurements at MR spectroscopy in patients with IDH wild-type glioblastoma. Materials and Methods This retrospective study was approved by the institutional review board, and informed consent was waived. Consecutive patients with histopathologically confirmed pre- and posttreatment glioblastoma were evaluated between December 2017 and August 2018. Spectroscopy parameters, including 2HG measurements, were obtained with single-voxel point-resolved spectroscopy, and apparent diffusion coefficient (ADC) values were calculated. Necrosis was graded according to the proportion of necrosis within a volume of interest. Poisson regression analyses were performed to determine factors related to false-positive 2HG measurements. Results A total of 82 patients were included (mean age, 55 years ± 12 [standard deviation]; 40 men). The 2HG measurement showed a false-positive rate of 21% (17 of 82; 95% CI: 13%, 31%) in patients with IDH wild-type glioblastoma. Multivariable analysis revealed that necrosis (prevalence ratio [PR], 3.9; 95% CI: 1.6, 9.4; P = .01) and ADC value (PR, 0.1 × 10-3 mm2/sec; 95% CI: [0.0, 0.7] × 10-3 mm2/sec; P = .02) were associated with a greater false-positive rate for the 2HG measurement. Necrosis of more than 20% was associated with a higher rate of false-positive 2HG measurements (50%) than was necrosis of 20% or less (15%, P = .01). The 2HG false-positive rate was higher in patients with pretreatment glioblastoma (46%) than in those with posttreatment glioblastoma (14%, P < .01). Among 17 patients with false-positive findings, 15 (88%; 95% CI: 64%, 99%) had a lactate concentration of 2.0 mmol/L or higher, and 14 (82%, 95% CI: 57%, 96%) had a lactate concentration of 3.0 mmol/L or higher. Conclusion Necrosis and apparent diffusion coefficient were associated with false-positive measurements of 2-hydroxyglutarate at MR spectroscopy in patients with isocitrate dehydrogenase wild-type glioblastoma. © RSNA, 2019 Online supplemental material is available for this article.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Imagem por Ressonância Magnética , Adulto , Idoso , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/epidemiologia , Neoplasias Encefálicas/patologia , Reações Falso-Positivas , Feminino , Glioblastoma/complicações , Glioblastoma/diagnóstico por imagem , Glioblastoma/epidemiologia , Glioblastoma/patologia , Glutaratos/química , Humanos , Isocitrato Desidrogenase/genética , Imagem por Ressonância Magnética/métodos , Imagem por Ressonância Magnética/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Necrose/diagnóstico por imagem , Necrose/etiologia , Necrose/patologia , Estudos Retrospectivos
17.
Mol Omics ; 15(3): 189-204, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31025681

RESUMO

Glutarylation, which is a newly identified posttranslational modification that occurs on lysine residues, has recently emerged as an important regulator of several metabolic and mitochondrial processes. However, the specific sites of modification on individual proteins, as well as the extent of glutarylation throughout the proteome, remain largely uncharacterized. Though informative, proteomic approaches based on mass spectrometry can be expensive, technically challenging and time-consuming. Therefore, the ability to predict glutarylation sites from protein primary sequences can complement proteomics analyses and help researchers study the characteristics and functional consequences of glutarylation. To this end, we used Random Forest (RF) machine learning strategies to identify the physiochemical and sequence-based features that correlated most substantially with glutarylation. We then used these features to develop a novel method to predict glutarylation sites from primary amino acid sequences using RF. Based on 10-fold cross-validation, the resulting algorithm, termed 'RF-GlutarySite', achieved efficiency scores of 75%, 81%, 68% and 0.50 with respect to accuracy (ACC), sensitivity (SN), specificity (SP) and Matthew's correlation coefficient (MCC), respectively. Likewise, using an independent test set, RF-GlutarySite exhibited ACC, SN, SP and MCC scores of 72%, 73%, 70% and 0.43, respectively. Results using both 10-fold cross validation and an independent test set were on par with or better than those achieved by existing glutarylation site predictors. Notably, RF-GlutarySite achieved the highest SN score among available glutarylation site prediction tools. Consequently, our method has the potential to uncover new glutarylation sites and to facilitate the discovery of relationships between glutarylation and well-known lysine modifications, such as acetylation, methylation and SUMOylation, as well as a number of recently identified lysine modifications, such as malonylation and succinylation.


Assuntos
Biologia Computacional/métodos , Glutaratos/metabolismo , Proteômica/métodos , Algoritmos , Sequência de Aminoácidos , Aminoácidos , Modelos Químicos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Máquina de Vetores de Suporte
18.
Acta Pharmacol Sin ; 40(10): 1292-1302, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31015738

RESUMO

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Glutaratos/farmacologia , Microglia/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/metabolismo , NF-kappa B/metabolismo , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
19.
Eur J Pharm Sci ; 133: 59-68, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30910648

RESUMO

Cocrystal formation may affect manufacturability (flow, compaction and processability) as well as solubility/dissolution, hygroscopicity and stability properties of drugs. Therefore, cocrystallization could be used to improve the pharmaceutical properties of low-soluble drugs such as ethenzamide. In this project, solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopy studies were performed for ethenzamide-glutaric acid to obtain more information about the ethenzamide cocrystallization process. The impact of the grinding time of the physical mixture (ethenzamide-glutaric acid) on cocrystal formation and the further spontaneous cocrystallization was evaluated using spectroscopic methods and curve-fitting analysis of the spectra. The influence of pressure on the yield of cocrystal formation was also described. Additionally, studies on the effect of magic-angle spinning during solid-state nuclear magnetic resonance spectra collection on the initiation of cocrystal formation, have been performed. Based on this research, conclusions regarding the influence of the different external factors, such as pressure during the tableting process and grinding time, on the cocrystal formation have been drawn for ethenzamide cocrystals.


Assuntos
Glutaratos/química , Salicilamidas/química , Cristalização , Composição de Medicamentos , Espectroscopia de Ressonância Magnética , Pressão , Espectroscopia de Infravermelho com Transformada de Fourier , Comprimidos
20.
Mater Sci Eng C Mater Biol Appl ; 98: 65-73, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30813069

RESUMO

Since Fe3O4 nanoparticles synthesized by plant extracts possess good bio-compatibility and superparamagnetic properties, the possibility of these could be used as a carrier in drug delivery. In this work, doxorubicin hydrochloride (DOX), an anti-cancer drug, loaded on glutaric anhydride-functionalized magnetic nanoparticles (Fe3O4@SiO2-Glu) was investigated at varying pH values for effective drug delivery. Various factors affecting the adsorption of DOX onto the Fe3O4@SiO2-Glu were examined, where the adsorption efficiency of DOX reached 92% at a concentration of 20 mg/L employing 10 mg of Fe3O4@SiO2-Glu at 303 K in pH 7.4. However, the adsorption efficiency of DOX was decreased to 18% at acidic pH value down to 3.0, implicating that the drug releasing process was controlled by pH. Adsorption kinetics was fitting to pseudo-second-order and the isothermal adsorption conformed to Freundlich isotherm. The morphology and surface composition of the synthesized Fe3O4@SiO2-Glu were characterized by SEM, TEM, and N2 adsorption/desorption isotherms, revealing that the specific surface area being 62.6 m2/g and the size ranging from ~30 to 50 nm. The zeta potential results indicated that Fe3O4@SiO2-Glu were negatively charged in various pH from 3 to 8.5. Characterizations by FTIR and UV-Vis techniques suggested that the DOX was absorbed and it can be delivered by Fe3O4@SiO2-Glu.


Assuntos
Anidridos/química , Doxorrubicina/química , Portadores de Fármacos/química , Glutaratos/química , Nanopartículas de Magnetita/química , Dióxido de Silício/química , Adsorção , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA