RESUMEN
MAF1 is a phosphoprotein that plays a critical role in cell growth control as the central regulator of RNA polymerase (Pol) III activity. Citrus MAF1 (CsMAF1) was identified as a direct target of PthA4, a bacterial effector protein required to induce tumors in citrus. CsMAF1 binds to Pol III to restrict transcription; however, exactly how CsMAF1 interacts with the polymerase and how phosphorylation modulates this interaction is unknown. Moreover, how CsMAF1 binds PthA4 is also obscure. Here we show that CsMAF1 binds predominantly to the WH1 domain of the citrus Pol III subunit C34 (CsC34) and that its phosphoregulatory region, comprising loop-3 and α-helix-2, contributes to this interaction. We also show that phosphorylation of this region decreases CsMAF1 affinity to CsC34, leading to Pol III derepression, and that Ser 45, found only in plant MAF1 proteins, is critical for CsC34 interaction and is phosphorylated by a new citrus AGC1 kinase. Additionally, we show that the C-terminal region of the citrus TFIIIB component BRF1 competes with CsMAF1 for CsC34 interaction, whereas the C-terminal region of CsMAF1 is essential for PthA4 binding. Based on CsMAF1 structural data, we propose a mechanism for how CsMAF1 represses Pol III transcription and how phosphorylation controls this process.
Asunto(s)
Citrus/genética , Proteínas de Plantas/metabolismo , ARN Polimerasa III/metabolismo , Citrus/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , ARN Polimerasa III/genética , Serina/metabolismo , Transcripción Genética , Levaduras/genéticaRESUMEN
The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , COVID-19 , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Humanos , Microscopía Electrónica , Simulación de Dinámica Molecular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , ARN Viral/genética , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
We postulated that Green tea (GT) improvements in non-alcoholic fatty liver disease (NAFLD) are dependent on adiponectin action in the liver. Male wild-type and adiponectin knockout (adipoKO) mice were induced to obesity for 8 weeks with a high-fat diet and then treated with GT for the last 12 weeks of the experimental protocol. Glucose and insulin tolerance tests, indirect calorimetry, histologic analysis of liver sections, and quantification of mRNA of hepatic genes related to glucose or fatty acid metabolism were performed. In vitro, we assessed the mechanism by which GT catechins act to improve hepatic steatosis by measuring lipid accumulation, and transcript levels of lipogenic genes in HepG2 cells treated with GT in the presence of a PPAR antagonist. Additionally, we performed a PPAR transactivation assay in 293T cells to test if catechins could activate PPARs. Different from wild-type mice, adipoKO animals treated with GT and fed a HFD gain body weight and fat mass, that were associated with a decrease in energy expenditure, were insulin resistant, and had no improvements in hepatic steatosis. Increased lipid levels were associated with no modulation of PPARα levels in the liver of adipoKO mice treated with GT. In vitro, we demonstrated GT catechins act to reduce hepatic steatosis in a PPARα-dependent manner, and especially epigallocatechin and epicatechin can indirectly activate PPARα, although it seems they are not direct ligands. By providing the mechanisms by which GT catechins act in the liver to improve steatosis, our data contribute to the discovery of novel therapeutic agents in the management of NAFLD.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , PPAR alfa , Adiponectina/metabolismo , Animales , Antioxidantes/metabolismo , Dieta Alta en Grasa/efectos adversos , Glucosa/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos , Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Té/químicaRESUMEN
The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Ligandos , Proteínas de la Nucleocápside/genética , ARN/metabolismo , Antivirales/farmacología , Unión ProteicaRESUMEN
Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present de novo splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation. By using a combination of biophysical assays and imaging approaches, we demonstrate that this mutant assembles solid-like condensates and amyloid-like fibrils. Although we observed greatly reduced expression of the mutant allele in a patient who exhibits skewed X inactivation, this appears to be enough to sequestrate healthy proteins into solid-like ectopic granules, compromising cell function. Therefore, our data suggest ID-linked DDX3X L556S mutation as a disorder arising from protein misfolding and aggregation.
RESUMEN
The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator's synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.
Asunto(s)
Resistencia a la Insulina/fisiología , PPAR gamma/metabolismo , Serina/metabolismo , Células 3T3 , Adipocitos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Células HEK293 , Humanos , Ratones , PPAR gamma/genética , Fosforilación/fisiología , Serina/genéticaRESUMEN
The potential contribution of green tea (GT) to the development of thermogenic/beige cells have been scarcely investigated. Here we investigated if the beneficial effects of GT in the induction of thermogenic/beige adipocytes results from an initial cell commitment during adipogenesis. Male C57Bl/6 mice (3 months) were divided into 3 groups: Control (chow diet), Obese (cafeteria diet), and Obese + GT. Mice received GT gavage (500 mg/kg of BW) over 12 weeks (5 days/week), after 4 weeks of diet, totalizing 16 weeks of experimentation. GT treatment increased energy expenditure (EE) in mice fed with cafeteria-diet leading to reduced BW gain, decreased adiposity, reduced inflammation, and improving insulin sensitivity. Those phenotypes were associated with enhanced expression of oxidative, thermogenic and beige genes. GT induced a futile cycle through de novo lipogenesis activating the thermogenic pathway. Induction of beige phenotype occurs autonomously in adipocytes and involves the PPARγ/FGF21/AMPK/UCP1 pathway. Our study identified that metabolic changes caused by GT may involve the temporal expression of PPARγ promoting the induction of thermogenic cells by reprogramming initial steps of adipocyte commitment.
Asunto(s)
Adipocitos Beige/efectos de los fármacos , Camellia sinensis/química , Obesidad/tratamiento farmacológico , Preparaciones de Plantas/administración & dosificación , Polifenoles/administración & dosificación , Termogénesis/efectos de los fármacos , Quinasas de la Proteína-Quinasa Activada por el AMP , Adipocitos Beige/citología , Adipocitos Beige/metabolismo , Adipogénesis/efectos de los fármacos , Animales , Metabolismo Energético/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatología , PPAR gamma/genética , PPAR gamma/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND AND OBJECTIVE: To determine factors causing inflammation after intravitreal aflibercept injections. PATIENTS AND METHODS: This case-control study included aflibercept-treated eyes with inflammation post-injection and aflibercept-treated control eyes. Medical records were analyzed to identify contributing factors. Biophysical tests were performed to characterize properties of particle aggregation. Institutional review board approval was obtained. RESULTS: Inflammation developed in six eyes; three patients had anterior uveitis, and five had vitreous cells. Oil droplets were seen in all cases. Saldanha Rodrigues (SR) syringes were used in all cases. Among controls, SR and Becton-Dickinson syringes were used in 10 and 17 eyes, respectively. Regression analysis showed an association between SR syringes and inflammation (odds ratio = 21.66; 95% confidence interval, 1.10-425.06; P = .043). Biophysical analyses primarily showed aggregation possibly from free oil droplets or protein-oil droplet aggregation. CONCLUSIONS: Post-injection inflammation was associated with SR syringes. Silicone oil droplets, especially after syringe agitation, might play a role in the inflammatory reaction. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:288-294.].
Asunto(s)
Receptores de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Enfermedades de la Retina/tratamiento farmacológico , Aceites de Silicona/efectos adversos , Uveítis/inducido químicamente , Estudios de Casos y Controles , Femenino , Humanos , Inyecciones Intravítreas , Masculino , Persona de Mediana Edad , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Microscopía con Lámpara de Hendidura , Jeringas , Uveítis/diagnóstico , Agudeza VisualRESUMEN
Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp90s are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp90s from different organisms have peculiarities despite their high sequence identity. Therefore, a detailed comparative analysis of several Hsp90 proteins is relevant to the overall understanding of their activity. Accordingly, the goal of this work was to evaluate the interaction of either ADP or ATP with recombinant Hsp90s from different organisms (human α and ß isoforms, Plasmodium falciparum, Leishmania braziliensis, yeast and sugarcane) by isothermal titration calorimetry. The measured thermodynamic signatures of those interactions indicated that despite the high identity among all Hsp90s, they have specific thermodynamic characteristics. Specifically, the interactions with ADP are driven by enthalpy but are opposed by entropy, whereas the interaction with ATP is driven by both enthalpy and entropy. Complimentary structural and molecular dynamics studies suggested that specific interactions with ADP that differ from those with ATP may contribute to the observed enthalpies and entropies. Altogether, the data suggest that selective inhibition may be more easily achieved using analogues of the Hsp90-ADP bound state than those of Hsp90-ATP bound state.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Secuencia de Aminoácidos , Proteínas HSP90 de Choque Térmico/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is considered a therapeutic target for metabolic disorders, cancer, and cardiovascular diseases. Here, we developed one pipeline for the screening of PPARß/δ agonists, which reduces the cost, time, and false-positive hits. The first step is an optimized 3-day long cellular transactivation assay based on reporter-gene technology, which is supported by automated liquid-handlers. This primary screening is followed by a confirmatory transactivation assay and by two biophysical validation methods (thermal shift assay (TSA) and (ANS) fluorescence quenching), which allow the calculation of the affinity constant, giving more information about the selected hits. All of the assays were validated using well-known commercial agonists providing trustworthy data. Furthermore, to validate and test this pipeline, we screened a natural extract library (560 extracts), and we found one plant extract that might be interesting for PPARß/δ modulation. In conclusion, our results suggested that we developed a cheaper and more robust pipeline that goes beyond the single activation screening, as it also evaluates PPARß/δ tertiary structure stabilization and the ligand affinity constant, selecting only molecules that directly bind to the receptor. Moreover, this approach might improve the effectiveness of the screening for agonists that target PPARß/δ for drug development.
RESUMEN
Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.
Asunto(s)
Nucleósidos/metabolismo , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Adenosina/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Citidina/metabolismo , Citosina/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Inosina/metabolismo , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Purina-Nucleósido Fosforilasa/genética , Schistosoma mansoni/química , Especificidad por SustratoRESUMEN
MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrus.