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1.
J Med Microbiol ; 70(4)2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33830911

RESUMEN

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa.Methodology. Computer-based virtual screening under consideration of the 'eNTRy' rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus.Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.


Asunto(s)
Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Transactivadores/antagonistas & inhibidores , Animales , Drosophila , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Simulación del Acoplamiento Molecular , Mutación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/mortalidad , Pseudomonas aeruginosa/genética , Tasa de Supervivencia , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Virulencia/efectos de los fármacos , Virulencia/genética
2.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33803640

RESUMEN

The LATS1 kinase has been described as a tumor suppressor in various cancers. However, its role in melanoma has not been fully elucidated. There are several processes involved in tumorigenesis, including melanin production. Melanin content positively correlates with the level of reactive oxygen species (ROS) inside the cell. Accordingly, the purpose of the study was to assess the role of LATS1 in melanogenesis and oxidative stress and its influence on tumor growth. We have knocked down LATS1 in primary melanocytes and melanoma cells and found that its expression is crucial for melanin synthesis, ROS production, and oxidative stress response. We showed that LATS1 ablation significantly decreased the melanogenesis markers' expression and melanin synthesis in melanocyte and melanoma cell lines. Moreover, silencing LATS1 resulted in enhanced oxidative stress. Reduced melanin content in LATS1 knocked down tumors was associated with increased tumor growth, pointing to melanin's protective role in this process. The study demonstrated that LATS1 is highly engaged in melanogenesis and oxidative stress control and affects melanoma growth. Our results may find the implications in the diagnosis and treatment of pigmentation disorders, including melanoma.


Asunto(s)
Melaninas/biosíntesis , Melanoma/patología , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Melanocitos/metabolismo , Melanoma/genética , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Hipoxia Tumoral/genética
3.
Life Sci ; 274: 119325, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33713665

RESUMEN

AIMS: The emergence of antibiotic tolerance was a tricky problem in the treatment of chronic Pseudomonas aeruginosa-infected cystic fibrosis and burn victims. The quorum sensing (QS) inhibitor may serve as a new tactic for the bacterial resistance by inhibiting the biofilm formation and the production of virulence factors. This study explored the potential of luteolin as a QS inhibitor against P. aeruginosa and the molecular mechanism involved. MAIN METHODS: Crystal violet staining, CLSM observation, and SEM analysis were carried out to assess the effect of luteolin on biofilm formation. The motility assays and the production of virulence factors were determined to evaluate the QS-inhibitory activity of luteolin. Acyl-homoserine lactone, RT-PCR, and molecular docking assays were conducted to explain its anti-QS mechanisms. KEY FINDINGS: The biofilm formation, the production of virulence factors, and the motility of P. aeruginosa could be efficiently inhibited by luteolin. Luteolin could also attenuate the accumulation of the QS-signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL) (P < 0.01) and downregulate the transcription levels of QS genes (lasR, lasI, rhlR, and rhlI) (P < 0.01). Molecular docking analysis indicated that luteolin had a greater docking affinity with LasR regulator protein compared with OdDHL. SIGNIFICANCE: This study is important as it reports the molecular mechanisms involved in the anti-biofilm formation activity of luteolin against P. aeruginosa. This study also indicated that luteolin could be helpful when used for the treatment of clinical drug-resistant infections of P. aeruginosa.


Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Luteolina/farmacología , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Transactivadores/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/antagonistas & inhibidores , Biopelículas/crecimiento & desarrollo , Homoserina/análogos & derivados , Homoserina/antagonistas & inhibidores , Humanos , Simulación del Acoplamiento Molecular , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Factores de Virulencia
5.
Mol Cell ; 81(8): 1682-1697.e7, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33651988

RESUMEN

The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control.


Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Acetilación , Animales , Células CHO , Proteína de Unión a CREB/metabolismo , Línea Celular , Cricetulus , Regulación de la Expresión Génica/genética , Células HEK293 , Histonas/metabolismo , Humanos , Cinética , Ratones , Transactivadores/metabolismo
6.
Nat Commun ; 12(1): 1703, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731717

RESUMEN

The factors regulating cellular identity are critical for understanding the transition from health to disease and responses to therapies. Recent literature suggests that autophagy compromise may cause opposite effects in different contexts by either activating or inhibiting YAP/TAZ co-transcriptional regulators of the Hippo pathway via unrelated mechanisms. Here, we confirm that autophagy perturbation in different cell types can cause opposite responses in growth-promoting oncogenic YAP/TAZ transcriptional signalling. These apparently contradictory responses can be resolved by a feedback loop where autophagy negatively regulates the levels of α-catenins, LC3-interacting proteins that inhibit YAP/TAZ, which, in turn, positively regulate autophagy. High basal levels of α-catenins enable autophagy induction to positively regulate YAP/TAZ, while low α-catenins cause YAP/TAZ activation upon autophagy inhibition. These data reveal how feedback loops enable post-transcriptional determination of cell identity and how levels of a single intermediary protein can dictate the direction of response to external or internal perturbations.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , alfa Catenina/metabolismo , Animales , Células Cultivadas , Células Epiteliales , Retroalimentación Fisiológica , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Transducción de Señal , alfa Catenina/química , alfa Catenina/genética
7.
Int J Mol Sci ; 22(4)2021 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-33668437

RESUMEN

Developmental growth and patterning are regulated by an interconnected signalling network of several pathways. In Drosophila, the Warts (Wts) kinase, a component of the Hippo signalling pathway, plays an essential role in regulating transcription and growth by phosphorylating its substrate Yorkie (Yki). The phosphorylation of Yki critically influences its localisation and activity as a transcriptional coactivator. In this study, we identified the homeodomain-interacting protein kinase (Hipk) as another kinase that phosphorylates Yki and mapped several sites of Yki phosphorylated by Hipk, using in vitro analysis: Ser168, Ser169/Ser172 and Ser255. These sites might provide auxiliary input for Yki regulation in vivo, as transgenic flies with mutations in these show prominent phenotypes; Hipk, therefore, represents an additional upstream regulator of Yki that works in concert with Wts.


Asunto(s)
Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Fosforilación/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transactivadores/genética
8.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671123

RESUMEN

The immune system constantly monitors the emergence of cancerous cells and eliminates them. CD8+ cytotoxic T lymphocytes (CTLs), which kill tumor cells and provide antitumor immunity, select their targets by recognizing tumor antigenic peptides presented by MHC class-I (MHC-I) molecules. Cancer cells circumvent immune surveillance using diverse strategies. A key mechanism of cancer immune evasion is downregulation of MHC-I and key proteins of the antigen processing and presentation machinery (APM). Even though impaired MHC-I expression in cancers is well-known, reversing the MHC-I defects remains the least advanced area of tumor immunology. The discoveries that NLRC5 is the key transcriptional activator of MHC-I and APM genes, and genetic lesions and epigenetic modifications of NLRC5 are the most common cause of MHC-I defects in cancers, have raised the hopes for restoring MHC-I expression. Here, we provide an overview of cancer immunity mediated by CD8+ T cells and the functions of NLRC5 in MHC-I antigen presentation pathways. We describe the impressive advances made in understanding the regulation of NLRC5 expression, the data supporting the antitumor functions of NLRC5 and a few reports that argue for a pro-tumorigenic role. Finally, we explore the possible avenues of exploiting NLRC5 for cancer immunotherapy.


Asunto(s)
Genes MHC Clase I , Inmunoterapia , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Transactivadores/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Humanos
9.
Nat Commun ; 12(1): 1846, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758180

RESUMEN

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.


Asunto(s)
Evolución Molecular Dirigida/métodos , Genes de Cambio , Ingeniería Genética/métodos , Técnicas Genéticas , Saccharomyces cerevisiae/genética , Biología Sintética/métodos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Basidiomycota/genética , Basidiomycota/metabolismo , Citometría de Flujo , Biblioteca de Genes , Genes Reporteros , Floroglucinol/análogos & derivados , Floroglucinol/farmacología , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Relación Señal-Ruido , Tetraciclina/farmacología , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , beta Caroteno/biosíntesis , beta Caroteno/genética , beta Caroteno/metabolismo
10.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540627

RESUMEN

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Proteínas Represoras/genética , Transactivadores/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Simulación por Computador , Homeostasis , Proteínas de Unión a Hierro/genética , Regiones Promotoras Genéticas , Receptores de Superficie Celular/genética , Yersinia enterocolitica/genética
11.
Science ; 371(6536): 1350-1355, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33632892

RESUMEN

Mitogens trigger cell division in animals. In plants, cytokinins, a group of phytohormones derived from adenine, stimulate cell proliferation. Cytokinin signaling is initiated by membrane-associated histidine kinase receptors and transduced through a phosphorelay system. We show that in the Arabidopsis shoot apical meristem (SAM), cytokinin regulates cell division by promoting nuclear shuttling of Myb-domain protein 3R4 (MYB3R4), a transcription factor that activates mitotic gene expression. Newly synthesized MYB3R4 protein resides predominantly in the cytoplasm. At the G2-to-M transition, rapid nuclear accumulation of MYB3R4-consistent with an associated transient peak in cytokinin concentration-feeds a positive feedback loop involving importins and initiates a transcriptional cascade that drives mitosis and cytokinesis. An engineered nuclear-restricted MYB3R4 mimics the cytokinin effects of enhanced cell proliferation and meristem growth.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , División Celular , Citocininas/metabolismo , Transactivadores/metabolismo , Transporte Activo de Núcleo Celular , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Puntos de Control del Ciclo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Carioferinas/metabolismo , Meristema/metabolismo , Mitosis/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal , Transactivadores/genética
12.
Nucleic Acids Res ; 49(5): 2740-2758, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33619536

RESUMEN

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Ratones , Invasividad Neoplásica , Neoplasias/metabolismo , Neoplasias/mortalidad , Transactivadores/metabolismo , Activación Transcripcional
13.
C R Biol ; 343(3): 247-255, 2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33621454

RESUMEN

The transcription of eukaryotic protein genes is controlled by a plethora of proteins which act together in multi-component complexes to facilitate the DNA dependent RNA polymerase II (Pol II) enzyme to bind to the transcription start site and to generate messenger RNA from the gene's coding sequence. The protein that guides the transcription machinery to the exact transcription start site is called TATA-box Binding Protein, or TBP. TBP is part of two large protein complexes involved in Pol II transcription, TFIID and SAGA. The two complexes share several subunits implicated in the interaction with TBP and contain proteins with structural elements highly homologous to nucleosomal histones. Despite the intensive study of transcription initiation, the mode of interaction of TBP with these complexes and its release upon DNA binding was elusive. In this study we demonstrate the quasi-atomic model of SAGA in complex with TBP. The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves a deformed octamer of histone-fold domains at the core of SAGA. This deformed octamer is precisely tuned to establish a peripheral site for TBP binding, where it is protected by steric hindrance against the binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires the general transcription factor TFIIA and whose efficiency correlates with the affinity of DNA to TBP.As the TBP binding machinery is highly similar in TFIID and SAGA, we demonstrated a universal mechanism of how TBP is delivered to gene promoters during transcription initiation.


Asunto(s)
Transactivadores , Factores de Transcripción , Organofosfatos , Regiones Promotoras Genéticas , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción Genética
14.
Int J Nanomedicine ; 16: 1021-1036, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33603366

RESUMEN

Purpose: To investigate the role and activation mechanism of TAZ in periodontal ligament stem cells (PDLSCs) perceiving hierarchical microgroove/nanopore topography. Materials and Methods: Titanium surface with hierarchical microgroove/nanopore topography fabricated by selective laser melting combined with alkali heat treatment (SLM-AHT) was used as experimental group, smooth titanium surface (Ti) and sandblasted, large-grit, acid-etched (SLA) titanium surface were employed as control groups. Alkaline phosphatase (ALP) activity assays, qRT-PCR, Western blotting, and immunofluorescence were carried out to evaluate the effect of SLM-AHT surface on PDLSC differentiation. Moreover, TAZ activation was investigated from the perspective of nuclear localization to transcriptional activity. TAZ knockdown PDLSCs were seeded on three titanium surfaces to detect osteogenesis- and adipogenesis-related gene expression levels. Immunofluorescence and Western blotting were employed to investigate the effect of the SLM-AHT surface on actin cytoskeletal polymerization and MAPK signaling pathway. Cytochalasin D and MAPK signaling pathway inhibitors were used to determine whether actin cytoskeletal polymerization and the MAPK signaling pathway were indispensable for TAZ activation. Results: Our results showed that SLM-AHT surface had a greater potential to promote PDLSC osteogenic differentiation while inhibiting adipogenic differentiation than the other two groups. The nuclear localization and transcriptional activity of TAZ were strongly enhanced on the SLM-AHT surface. Moreover, after TAZ knockdown, the enhanced osteogenesis and decreased adipogenesis in SLM-AHT group could not be observed. In addition, SLM-AHT surface could promote actin cytoskeletal polymerization and upregulate p-ERK and p-p38 protein levels. After treatment with cytochalasin D and MAPK signaling pathway inhibitors, differences in the TAZ subcellular localization and transcriptional activity were no longer observed among the different titanium surfaces. Conclusion: Our results demonstrated that actin cytoskeletal polymerization and MAPK signaling pathway activation triggered by SLM-AHT surface were essential for TAZ activation, which played a dominant role in SLM-AHT surface-induced stem cell fate decision.


Asunto(s)
Diferenciación Celular , Nanoporos , Células Madre/citología , Transactivadores/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Adipogénesis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Osteogénesis/efectos de los fármacos , Ligamento Periodontal/citología , Polimerizacion , Propiedades de Superficie , Titanio/farmacología
15.
Am J Physiol Heart Circ Physiol ; 320(3): H999-H1016, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33416454

RESUMEN

We aimed to determine 1) the mechanism(s) that enables glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)-driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs, and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC-restricted genes. G6PD knockdown or inhibition decreased (48.5%) histone deacetylase (HDAC) activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control Sprague-Dawley (SD) rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4 mo increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate that G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.NEW & NOTEWORTHY This study gives detailed mechanistic insight about the regulation of smooth muscle cell (SMC) phenotype by metabolic reprogramming and glucose-6-phosphate dehydrogenase (G6PD) in diabetes and metabolic syndrome. We demonstrate that G6PD controls the chromatin modifications by regulating histone deacetylase (HDAC) activity, which deacetylates histone 3-lysine 9 and 27. Notably, inhibition of G6PD decreases HDAC activity and enriches H3K27ac on myocardin gene promoter to enhance the expression of SMC-restricted genes. Also, we demonstrate for the first time that G6PD inhibitor treatment accentuates metabolic and transcriptomic reprogramming to reduce neointimal formation in coronary artery and large artery elastance in metabolic syndrome rats.


Asunto(s)
Glucosafosfato Deshidrogenasa/metabolismo , Histonas/metabolismo , Síndrome Metabólico/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Glucosafosfato Deshidrogenasa/genética , Hemodinámica , Humanos , Masculino , Síndrome Metabólico/genética , Síndrome Metabólico/patología , Síndrome Metabólico/fisiopatología , Ratones Transgénicos , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Mutación , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas Sprague-Dawley , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Remodelación Vascular
16.
Arterioscler Thromb Vasc Biol ; 41(3): 1105-1123, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33406884

RESUMEN

OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.


Asunto(s)
Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Factores de Transcripción MEF2/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Endotelio Vascular/patología , Femenino , Regulación de la Expresión Génica , Homeostasis , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción MEF2/deficiencia , Factores de Transcripción MEF2/genética , Masculino , Ratones , Ratones Noqueados , Receptores Notch/genética , Transducción de Señal , Transactivadores/metabolismo
17.
Cell Mol Life Sci ; 78(7): 3525-3542, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33469705

RESUMEN

Metastasis Associated in Colon Cancer 1 (MACC1) is a novel prognostic, predictive and causal biomarker for tumor progression and metastasis in many cancer types, including colorectal cancer. Besides its clinical value, little is known about its molecular function. Its similarity to SH3BP4, involved in regulating uptake and recycling of transmembrane receptors, suggests a role of MACC1 in endocytosis. By exploring the MACC1 interactome, we identified the clathrin-mediated endocytosis (CME)-associated proteins CLTC, DNM2 and AP-2 as MACC1 binding partners. We unveiled a MACC1-dependent routing of internalized transferrin receptor towards recycling. Elevated MACC1 expression caused also the activation and internalization of EGFR, a higher rate of receptor recycling, as well as earlier and stronger receptor activation and downstream signaling. These effects are limited by deletion of CME-related protein interaction sites in MACC1. Thus, MACC1 regulates CME and receptor recycling, causing increased growth factor-mediated downstream signaling and cell proliferation. This novel mechanism unveils potential therapeutic intervention points restricting MACC1-driven metastasis.


Asunto(s)
Clatrina/metabolismo , Neoplasias Colorrectales/patología , Endocitosis , Regulación Neoplásica de la Expresión Génica , Receptores de Transferrina/metabolismo , Transactivadores/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Ratones , Proteoma/análisis , Proteoma/metabolismo , Receptores de Transferrina/genética , Transactivadores/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
18.
Dev Cell ; 56(1): 81-94.e10, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33400912

RESUMEN

Homeostasis of intestinal epithelia is maintained by coordination of the proper rate of regeneration by stem cell division with the rate of cell loss. Regeneration of host epithelia is normally quiescent upon colonization of commensal bacteria; however, the epithelia often develop dysplasia in a context-dependent manner, the cause and underlying mechanism of which remain unclear. Here, we show that in Drosophila intestine, autophagy lowers the sensitivity of differentiated enterocytes to reactive oxygen species (ROS) that are produced in response to commensal bacteria. We find that autophagy deficiency provokes ROS-dependent excessive regeneration and subsequent epithelial dysplasia and barrier dysfunction. Mechanistically, autophagic substrate Ref(2)P/p62, which co-localizes and physically interacts with Dachs, a Hippo signaling regulator, accumulates upon autophagy deficiency and thus inactivates Hippo signaling, resulting in stem cell over-proliferation non-cell autonomously. Our findings uncover a mechanism whereby suppression of undesirable regeneration by autophagy maintains long-term homeostasis of intestinal epithelia.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismo , Transactivadores/metabolismo , Envejecimiento/metabolismo , Animales , Animales Modificados Genéticamente , Autofagia/efectos de los fármacos , Autofagia/genética , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Sulfato de Dextran/toxicidad , Drosophila/genética , Drosophila/inmunología , Drosophila/fisiología , Proteínas de Drosophila/genética , Enterocitos/metabolismo , Técnicas de Silenciamiento del Gen , Estudio de Asociación del Genoma Completo , Homeostasis , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Intestinos/citología , Intestinos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Musculares , Miosinas/genética , Miosinas/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
19.
Mol Cell ; 81(3): 571-583.e6, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33412111

RESUMEN

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriófagos/metabolismo , Pseudomonas aeruginosa/metabolismo , Percepción de Quorum , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Proteínas Bacterianas/genética , Bacteriófagos/genética , Bacteriófagos/patogenicidad , Fimbrias Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Piocianina/metabolismo , Transactivadores/genética , Proteínas Virales/genética
20.
Cell Prolif ; 54(2): e12987, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33415745

RESUMEN

OBJECTIVES: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells. MATERIAL AND METHODS: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues. RESULTS: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin. CONCLUSIONS: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.


Asunto(s)
Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Estrés Mecánico , Actinas/metabolismo , Actomiosina/metabolismo , Células Cultivadas , Senescencia Celular , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Metaloproteinasa 3 de la Matriz/metabolismo , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Núcleo Pulposo/citología , Núcleo Pulposo/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transactivadores/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismo
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