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1.
PLoS Genet ; 19(2): e1010618, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36735730

RESUMEN

Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis, de-repression of pervasive transcription by inactivation of rho revealed the role of Rho in the regulation of post-exponential differentiation programs. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain (Rho+) that expresses rho at a relatively stable high level in order to compensate for its decrease in the wild-type cells entering stationary phase. The RNAseq analysis of Rho+, WT and Δrho strains (expression profiles can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/) shows that Rho over-production enhances the termination efficiency of Rho-sensitive terminators, thus reducing transcriptional read-through and antisense transcription genome-wide. Moreover, the Rho+ strain exhibits global alterations of sense transcription with the most significant changes observed for the AbrB, CodY, and stringent response regulons, forming the pathways governing the transition to stationary phase. Subsequent physiological analyses demonstrated that maintaining rho expression at a stable elevated level modifies stationary phase-specific physiology of B. subtilis cells, weakens stringent response, and thereby negatively affects the cellular adaptation to nutrient limitations and other stresses, and blocks the development of genetic competence and sporulation. These results highlight the Rho-specific termination of transcription as a novel element controlling stationary phase. The release of this control by decreasing Rho levels during the transition to stationary phase appears crucial for the functionality of complex gene networks ensuring B. subtilis survival in stationary phase.


Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Ciclo Celular , Regulación Bacteriana de la Expresión Génica/genética
2.
Biochem Biophys Res Commun ; 695: 149400, 2024 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-38160530

RESUMEN

SETD2 (SET-domain containing protein 2) is a histone methyltransferase (HMT) of the SET family responsible for the trimethylation of K36 of histone H3, thus producing the epigenetic mark H3K36me3. Recent studies have shown that certain SET family HMTs, such as SMYD2, SMYD3 or SETDB1 can also methylate protein kinases and therefore be involved in signaling pathways. Here we provide structural and enzymatic evidence showing that SETD2 methylates the protein tyrosine kinase ACK1 in vitro. ACK1 is recognized as a major integrator of signaling from various receptor tyrosine kinases. Using ACK1 peptides and recombinant proteins, we show that SETD2 methylates the K514 residue of ACK1 generating K514 mono, di or tri-methylation. Interestingly, K514 is found in a "H3K36-like" motif of ACK1 which is known to be post-translationally modified and to be involved in protein-protein interaction. The crystal structure of SETD2 catalytic domain in complex with an ACK1 peptide further provides the structural basis for the methylation of ACK1 K514 by SETD2. Our work therefore strongly suggests that ACK1 could be a novel non-histone substrate of SETD2 and further supports that SET HMTs, such as SETD2, could be involved in both epigenetic regulations and cell signaling.


Asunto(s)
Histonas , Proteínas Tirosina Quinasas , Proteínas Tirosina Quinasas/metabolismo , Histonas/metabolismo , Metilación , N-Metiltransferasa de Histona-Lisina/genética , Procesamiento Proteico-Postraduccional
3.
Cell Mol Life Sci ; 80(12): 370, 2023 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-37989807

RESUMEN

Individuals with Down syndrome (DS) have a higher prevalence of obesity compared to the general population. Conventionally, this has been attributed to endocrine issues and lack of exercise. However, deficits in neural reward responses and dopaminergic disturbances in DS may be contributing factors. To investigate this, we focused on a mouse model (Ts65Dn) bearing some triplicated genes homologous to trisomy 21. Through detailed meal pattern analysis in male Ts65Dn mice, we observed an increased preference for energy-dense food, pointing towards a potential "hedonic" overeating behavior. Moreover, trisomic mice exhibited higher scores in compulsivity and inflexibility tests when limited access to energy-dense food and quinine hydrochloride adulteration were introduced, compared to euploid controls. Interestingly, when we activated prelimbic-to-nucleus accumbens projections in Ts65Dn male mice using a chemogenetic approach, impulsive and compulsive behaviors significantly decreased, shedding light on a promising intervention avenue. Our findings uncover a novel mechanism behind the vulnerability to overeating and offer potential new pathways for tackling obesity through innovative interventions.


Asunto(s)
Síndrome de Down , Trisomía , Humanos , Masculino , Ratones , Animales , Síndrome de Down/genética , Modelos Animales de Enfermedad , Corteza Prefrontal , Hiperfagia/genética , Obesidad/genética
4.
Nucleic Acids Res ; 50(10): 5545-5564, 2022 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-35609998

RESUMEN

The aryl hydrocarbon receptor (AHR) regulates the expression of numerous genes in response to activation by agonists including xenobiotics. Although it is well appreciated that environmental signals and cell intrinsic features may modulate this transcriptional response, how it is mechanistically achieved remains poorly understood. We show that hexokinase 2 (HK2) a metabolic enzyme fuelling cancer cell growth, is a transcriptional target of AHR as well as a modulator of its activity. Expression of HK2 is positively regulated by AHR upon exposure to agonists both in human cells and in mice lung tissues. Conversely, over-expression of HK2 regulates the abundance of many proteins involved in the regulation of AHR signalling and these changes are linked with altered AHR expression levels and transcriptional activity. HK2 expression also shows a negative correlation with AHR promoter methylation in tumours, and these tumours with high HK2 expression and low AHR methylation are associated with a worse overall survival in patients. In sum, our study provides novel insights into how AHR signalling is regulated which may help our understanding of the context-specific effects of this pathway and may have implications in cancer.


Asunto(s)
Hexoquinasa , Receptores de Hidrocarburo de Aril , Animales , Hexoquinasa/genética , Hexoquinasa/metabolismo , Hexoquinasa/farmacología , Humanos , Ratones , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Xenobióticos
5.
Genet Med ; 24(10): 2004-2013, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35951014

RESUMEN

PURPOSE: Although some caregivers are using epigallocatechin gallate (EGCG) off label in hopes of improving cognition in young adults with Down syndrome (DS), nothing is known about its safety, tolerability, and efficacy in the DS pediatric population. We aimed to evaluate safety and tolerability of a dietary supplement containing EGCG and if EGCG improves cognitive and functional performance. METHODS: A total of 73 children with DS (aged 6-12 years) were randomized. Participants received 0.5% EGCG (10 mg/kg daily dose) or placebo for 6 months with 3 months follow up after treatment discontinuation. RESULTS: In total, 72 children were treated and 66 completed the study. A total of 38 participants were included in the EGCG group and 35 in the placebo group. Of 72 treated participants, 62 (86%) had 229 treatment-emergent adverse events (AEs). Of 37 participants in the EGCG group, 13 (35%) had 18 drug-related treatment-emergent AEs and 12 of 35 (34%) from the placebo group had 22 events. In the EGCG group, neither severe AEs nor increase in the incidence of AEs related to safety biomarkers were observed. Cognition and functionality were not improved compared with placebo. Secondary efficacy outcomes in girls point to a need for future work. CONCLUSION: The use of EGCG is safe and well-tolerated in children with DS, but efficacy results do not support its use in this population.


Asunto(s)
Catequina , Síndrome de Down , Catequina/efectos adversos , Catequina/análogos & derivados , Niño , Cognición , Suplementos Dietéticos , Método Doble Ciego , Síndrome de Down/tratamiento farmacológico , Femenino , Humanos , Masculino
6.
Diabetologia ; 63(2): 395-409, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31796987

RESUMEN

AIMS/HYPOTHESIS: During the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-ßH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment. METHODS: EndoC-ßH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity. RESULTS: EndoC-ßH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-ßH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion. CONCLUSIONS/INTERPRETATION: The present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity. DATA AVAILABILITY: Microarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code.


Asunto(s)
Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ácido Palmítico/farmacología , Estearoil-CoA Desaturasa/metabolismo , Apoptosis/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Secreción de Insulina/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción HES-1/metabolismo
7.
Int J Mol Sci ; 21(4)2020 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-32092951

RESUMEN

Epigallocatechin gallate (EGCG) is an inhibitor of DYRK1A, a serine/threonine kinase considered to be a major contributor of cognitive dysfunctions in Down syndrome (DS). Two clinical trials in adult patients with DS have shown the safety and efficacy to improve cognitive phenotypes using commercial green tea extract containing EGCG (45% content). In the present study, we performed a preclinical study using FontUp®, a new nutritional supplement with a chocolate taste specifically formulated for the nutritional needs of patients with DS and enriched with a standardized amount of EGCG in young mice overexpressing Dyrk1A (TgBACDyrk1A). This preparation is differential with previous one used, because its green tea extract has been purified to up 94% EGCG of total catechins. We analyzed the in vitro effect of green tea catechins not only for EGCG, but for others residually contained in FontUp®, on DYRK1A kinase activity. Like EGCG, epicatechin gallate was a noncompetitive inhibitor against ATP, molecular docking computations confirming these results. Oral FontUp® normalized brain and plasma biomarkers deregulated in TgBACDyrk1A, without negative effect on liver and cardiac functions. We compared the bioavailability of EGCG in plasma and brain of mice and have demonstrated that EGCG had well crossed the blood-brain barrier.


Asunto(s)
Encéfalo/efectos de los fármacos , Catequina/análogos & derivados , Síndrome de Down/dietoterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Té/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Disponibilidad Biológica , Biomarcadores/sangre , Biomarcadores/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Catequina/administración & dosificación , Catequina/efectos adversos , Catequina/química , Catequina/uso terapéutico , Suplementos Dietéticos , Síndrome de Down/sangre , Síndrome de Down/enzimología , Síndrome de Down/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/uso terapéutico , Polifenoles/análisis , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Regulación hacia Arriba , Quinasas DyrK
8.
Neurobiol Dis ; 129: 13-28, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31051234

RESUMEN

Finding new targets to control or reduce seizure activity is essential to improve the management of epileptic patients. We hypothesized that activation of the pre-synaptic and inhibitory metabotropic glutamate receptor type 7 (mGlu7) reduces spontaneous seizures. We tested LSP2-9166, a recently developed mGlu7/4 agonist with unprecedented potency on mGlu7 receptors, in two paradigms of epileptogenesis. In a model of chemically induced epileptogenesis (pentylenetetrazole systemic injection), LSP2-9166 induces an anti-epileptogenic effect rarely observed in preclinical studies. In particular, we found a bidirectional modulation of seizure progression by mGlu4 and mGlu7 receptors, the latter preventing kindling. In the intra-hippocampal injection of kainic acid mouse model that mimics the human mesial temporal lobe epilepsy, we found that LSP2-9166 reduces seizure frequency and hippocampal sclerosis. LSP2-9166 also acts as an anti-seizure drug on established seizures in both models tested. Specific modulation of the mGlu7 receptor could represent a novel approach to reduce pathological network remodeling.


Asunto(s)
Aminobutiratos/farmacología , Anticonvulsivantes/farmacología , Hipocampo/efectos de los fármacos , Receptores de Glutamato Metabotrópico/agonistas , Convulsiones/metabolismo , Animales , Epilepsia/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/metabolismo , Excitación Neurológica/efectos de los fármacos , Ratones , Ratones Mutantes
9.
J Pharmacol Exp Ther ; 369(3): 489-502, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30940696

RESUMEN

Methionine deprivation induces growth arrest and death of cancer cells. To eliminate l-methionine we produced, purified, and characterized the recombinant pyridoxal 5'-phosphate (PLP)-dependent l-methionine γ-lyase (MGL)- BL929 from the cheese-ripening Brevibacterium aurantiacum Transformation of an Escherichia coli strain with the gene BL929 from B. aurantiacum optimized for E. coli expression led to production of the MGL-BL929. Elimination of l-methionine and cytotoxicity in vitro were assessed, and methylation-sensitive epigenetics was explored for changes resulting from exposure of cancer cells to the enzyme. A bioreactor was built by encapsulation of the protein in human erythrocytes to achieve sustained elimination of l-methionine in extracellular fluids. Catalysis was limited to α,γ-elimination of l-methionine and l-homocysteine. The enzyme had no activity on other sulfur-containing amino acids. Enzyme activity decreased in presence of serum albumin or plasma resulting from reduction of PLP availability. Elimination of l-methionine induced cytotoxicity on a vast panel of human cancer cell lines and spared normal cells. Exposure of colorectal carcinoma cells to the MGL-BL929 reduced methyl-CpG levels of hypermethylated gene promoters including that of CDKN2A, whose mRNA expression was increased, together with a decrease in global histone H3 dimethyl lysine 9. The MGL-erythrocyte bioreactor durably preserves enzyme activity in vitro and strongly eliminates l-methionine from medium.


Asunto(s)
Brevibacterium/enzimología , Liasas de Carbono-Azufre/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Metionina/metabolismo , Proteínas Recombinantes/farmacología , Adulto , Animales , Reactores Biológicos , Cápsulas , Línea Celular Tumoral , Humanos , Ratones
10.
Biochem Biophys Res Commun ; 503(2): 703-709, 2018 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-29932913

RESUMEN

We reported recently that the Parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO and YajL function as deglycases that repair proteins and nucleotides from endogeneous glycation by glyoxal and methylglyoxal, two reactive by-products of glucose metabolism responsible for up to 60% of glycation damage. Here, we show that DJ-1, deglycase 1 and deglycase 2 repair glyoxal- and methylglyoxal-glycated substrates, whereas deglycase 3 principally repairs glyoxal-glycated substrates. Moreover, deglycase 1 and 2 are overexpressed in stationary phase, whereas deglycase 3 is steadily expressed throughout bacterial growth. Finally, deglycase mutants overexpress glyoxalases, aldoketoreductases, glutathione-S-transferase and efflux pumps to alleviate carbonyl stress. In the discussion, we present an overview of the multiple functions of DJ-1 proteins. Our thourough work on deglycases provides compelling evidence that their previously reported glyoxalase III activity merely reflects their deglycase activity. Moreover, for their deglycase activity the Maillard deglycases likely recruit: i) their chaperone activity to interact with glycated proteins, ii) glyoxalase 1 activity to catalyze the rearrangement of Maillard products (aminocarbinols and hemithioacetals) into amides and thioesters, respectively, iii) their protease activity to cleave amide bonds of glycated arginine, lysine and guanine, and iv) glyoxalase 2 activity to cleave thioester bonds of glycated cysteine. Finally, because glycation affects many cellular processes, the discovery of the Maillard deglycases, awaited since 1912, likely constitutes a major advance for medical research, including ageing, cancer, atherosclerosis, neurodegenerative, post-diabetic, renal and autoimmune diseases.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Proteínas Ribosómicas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Glioxal/metabolismo , Humanos , Piruvaldehído/metabolismo
11.
J Pharmacol Exp Ther ; 366(2): 238-243, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29858389

RESUMEN

The current study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (H4PteGlu) into methylene tetra hydro pteroylglutamate (CH2-H4PteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyzes formation of CH2-H4PteGlu by transfer of the Cß of serine to H4PteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The median-effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29 and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the KD for binding to SHMT, and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.


Asunto(s)
Antineoplásicos/farmacología , Fluorouracilo/farmacología , Ácido Fólico/farmacología , Leucovorina/farmacología , Fosfato de Piridoxal/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Sinergismo Farmacológico , Células HCT116 , Células HT29 , Humanos , Concentración 50 Inhibidora , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ratones
12.
PLoS Genet ; 11(7): e1005384, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26181065

RESUMEN

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.


Asunto(s)
Síndrome de Bloom/genética , Citidina Desaminasa/genética , Poli(ADP-Ribosa) Polimerasas/genética , Pirimidinas/metabolismo , Síndrome de Bloom/patología , Línea Celular , Centrómero/genética , Sitios Frágiles del Cromosoma/genética , Segregación Cromosómica/genética , Citidina Desaminasa/deficiencia , Replicación del ADN/genética , Inestabilidad Genómica , Humanos , Mitosis/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/biosíntesis , RecQ Helicasas/genética , Intercambio de Cromátides Hermanas/genética
13.
Artículo en Inglés | MEDLINE | ID: mdl-28807907

RESUMEN

De novo pyrimidine biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1H-indol-3-yl)-2,3-dihydro-4H-furo[3,2-c]chromen-4-one derivatives as a new class of pyrimidine biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo pyrimidine biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of pyrimidine biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit pyrimidine biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.


Asunto(s)
Antivirales/farmacología , Virus Chikungunya/inmunología , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Interferón Tipo I/biosíntesis , Interferones/biosíntesis , Virus del Sarampión/inmunología , Pirimidinas/biosíntesis , Antivirales/química , Línea Celular , Cromonas/química , Dicumarol/farmacología , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Inmunidad Innata/inmunología , Indoles/química , Interferón Tipo I/inmunología , Interferones/inmunología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Relación Estructura-Actividad , Interferón lambda
14.
Biochem Biophys Res Commun ; 483(1): 387-391, 2017 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-28013050

RESUMEN

We discovered recently that Parkinsonism-associated DJ-1 and its bacterial homologs function as protein deglycases that repair glyoxal- and methylglyoxal-glycated proteins. Protein glycation levels are 2- to 10-fold increased in deglycase-depleted cells, and deglycase mutants display up to 500-fold loss of viability in methylglyoxal or glucose-containing media, suggesting that these deglycases play important roles in protecting cells against electrophile and carbonyl stress. Although the deglycase activity of DJ-1 is well supported by extensive biochemical work, Pfaff et al. (J. Biol. Chem. in presshttp://dx.doi.org/10.1074/jbc.M116.743823) claimed in a recent study that deglycation of the hemithioacetal formed upon cysteine glycation by methylglyoxal results from a Tris buffer artefact. Here, we show that this is not the case, and that DJ-1 and its homologs are the bona fide deglycases awaited since the Maillard discovery.


Asunto(s)
Proteínas Oncogénicas/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Acetilcisteína/química , Artefactos , Medios de Cultivo/química , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Glucosa/química , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Glioxal/química , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Trastornos Parkinsonianos/metabolismo , Piruvaldehído/metabolismo
15.
Biochim Biophys Acta Gen Subj ; 1861(1 Pt A): 3144-3153, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27456766

RESUMEN

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine. METHODS: We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS. RESULTS: The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate FeIII state. Imidazole (Im) derivatives, such as miconazole, acted as FeIII ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model. MAJOR CONCLUSION AND GENERAL SIGNIFICANCE: Our study describes for the first time the mode of interaction of several FeIII-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.


Asunto(s)
Familia 2 del Citocromo P450/química , Familia 2 del Citocromo P450/metabolismo , Modelos Moleculares , Ácido Araquidónico/química , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/química , Ácidos Araquidónicos/metabolismo , Biocatálisis , Debrisoquina/química , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Humanos , Imidazoles/química , Ácidos Láuricos/química , Ligandos , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Unión Proteica , Piridinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Espectrofotometría Ultravioleta , Especificidad por Sustrato
16.
J Biol Chem ; 290(3): 1885-97, 2015 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-25416785

RESUMEN

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.


Asunto(s)
Arginina/química , Cisteína/química , Glioxal/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisina/química , Proteínas Oncogénicas/metabolismo , Trastornos Parkinsonianos/metabolismo , Piruvaldehído/química , Acetilcisteína/química , Albúminas/química , Apoptosis , Aspartato Aminotransferasas/metabolismo , Catálisis , Supervivencia Celular , Escherichia coli/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa/química , Glicolatos/química , Humanos , Lactatos/química , Espectrometría de Masas , Estrés Oxidativo , Proteína Desglicasa DJ-1
17.
EMBO J ; 31(22): 4276-88, 2012 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-22990237

RESUMEN

Obesity-related diseases such as diabetes and dyslipidemia result from metabolic alterations including the defective conversion, storage and utilization of nutrients, but the central mechanisms that regulate this process of nutrient partitioning remain elusive. As positive regulators of feeding behaviour, agouti-related protein (AgRP) producing neurons are indispensible for the hypothalamic integration of energy balance. Here, we demonstrate a role for AgRP-neurons in the control of nutrient partitioning. We report that ablation of AgRP-neurons leads to a change in autonomic output onto liver, muscle and pancreas affecting the relative balance between lipids and carbohydrates metabolism. As a consequence, mice lacking AgRP-neurons become obese and hyperinsulinemic on regular chow but display reduced body weight gain and paradoxical improvement in glucose tolerance on high-fat diet. These results provide a direct demonstration of a role for AgRP-neurons in the coordination of efferent organ activity and nutrient partitioning, providing a mechanistic link between obesity and obesity-related disorders.


Asunto(s)
Proteína Relacionada con Agouti/metabolismo , Hipotálamo/metabolismo , Animales , Metabolismo de los Hidratos de Carbono/fisiología , Ingestión de Alimentos/fisiología , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Neuronas/metabolismo , Obesidad/metabolismo , Páncreas/metabolismo , Aumento de Peso/fisiología
18.
PLoS Pathog ; 10(1): e1003893, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24453979

RESUMEN

Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle.


Asunto(s)
Ciclo del Ácido Cítrico , Francisella tularensis/metabolismo , Ácido Glutámico/metabolismo , Macrófagos/microbiología , Fagosomas/metabolismo , Tularemia/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Femenino , Francisella tularensis/genética , Francisella tularensis/patogenicidad , Ácido Glutámico/genética , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fagosomas/genética , Fagosomas/microbiología , Fagosomas/patología , Tularemia/genética
19.
Infect Immun ; 83(1): 173-83, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25332124

RESUMEN

Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens.


Asunto(s)
Adaptación Fisiológica , Francisella tularensis/fisiología , Isoleucina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Citosol/microbiología , Modelos Animales de Enfermedad , Femenino , Francisella tularensis/genética , Francisella tularensis/crecimiento & desarrollo , Francisella tularensis/metabolismo , Eliminación de Gen , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos BALB C , Fagosomas/microbiología , Tularemia/microbiología , Tularemia/patología
20.
Biochem Biophys Res Commun ; 463(4): 1305-10, 2015 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-26102038

RESUMEN

Hsp31 belongs to the PfpI/Hsp31/DJ-1 superfamily, and has been reported to display chaperone, peptidase and glutathione-independent glyoxalase activities. Here, we show that Hsp31 repairs glyoxal- and methylglyoxal-glycated amino acids and proteins and releases repaired proteins and lactate or glycolate, respectively. Hsp31 deglycates cysteine, arginine and lysine by acting on early glycation intermediates (hemithioacetals and aminocarbinols) and prevents the formation of Schiff bases and advanced glycation endproducts. Hsp31 repairs glycated serum albumin, glyceraldehyde-3-phosphate dehydrogenase, fructose biphosphate aldolase and aspartate aminotransferase. Moreover, we show that bacterial extracts from the hchA mutant display increased glycation levels and that the apparent glyoxalase activity of Hsp31 reflects its deglycase activity. Our results suggest that other Hsp31 members, previously characterized as glutathione-independent glyoxalases, likely function as protein deglycases.


Asunto(s)
Proteínas de Escherichia coli/fisiología , Glioxal/farmacología , Chaperonas Moleculares/fisiología , Piruvaldehído/farmacología , Arginina/metabolismo , Cisteína/metabolismo , Glucosa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Lisina/metabolismo , Bases de Schiff
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