RESUMEN
Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gluconeogénesis/efectos de los fármacos , Hipoglucemiantes/administración & dosificación , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/antagonistas & inhibidores , Acetilación , Animales , Glucemia/metabolismo , Células Cultivadas , Glucosa/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Resistencia a la Insulina , Ratones , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.
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Gluconeogénesis , Glucosa/deficiencia , Histonas/metabolismo , Metabolismo de los Lípidos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Metabolismo de los Lípidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.
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Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , Replicación del ADN/genética , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Nucleosomas/genética , RecQ Helicasas/genética , Saccharomyces cerevisiae/genética , CohesinasRESUMEN
The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.
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Poro Nuclear/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/metabolismo , Activación Transcripcional , Animales , Línea Celular , Células Cultivadas , Cromatina/metabolismo , Humanos , Ratones , Ratones Noqueados , Mitógenos/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-myc/química , Serina/metabolismo , Cicatrización de Heridas , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The transcriptional coactivators Mediator and two histone acetyltransferase (HAT) complexes, NuA4 and SAGA, play global roles in transcriptional activation. Here we explore the relative contributions of these factors to RNA polymerase II association at specific genes and gene classes by rapid nuclear depletion of key complex subunits. We show that the NuA4 HAT Esa1 differentially affects certain groups of genes, whereas the SAGA HAT Gcn5 has a weaker but more uniform effect. Relative dependence on Esa1 and Tra1, a shared component of NuA4 and SAGA, distinguishes two large groups of coregulated growth-promoting genes. In contrast, we show that the activity of Mediator is particularly important at a separate, small set of highly transcribed TATA-box-containing genes. Our analysis indicates that at least three distinct combinations of coactivator deployment are used to generate moderate or high transcription levels and suggests that each may be associated with distinct forms of regulation.
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Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/fisiología , Complejo Mediador/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Activación Transcripcional , Acetilación , Histonas/metabolismo , Complejo Mediador/metabolismo , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Transcripción GenéticaRESUMEN
Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.
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Transporte Activo de Núcleo Celular , Antígenos de Histocompatibilidad Clase I , Péptidos y Proteínas de Señalización Intracelular , Activación Transcripcional , Humanos , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Células MCF-7 , Factores de Transcripción p300-CBP/metabolismo , Factores de Transcripción p300-CBP/genéticaRESUMEN
Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.
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Ensamble y Desensamble de Cromatina , Eucromatina/enzimología , Silenciador del Gen , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Acetilación , Eucromatina/genética , Retroalimentación Fisiológica , Regulación Fúngica de la Expresión Génica , Heterocromatina/enzimología , Heterocromatina/genética , Histona Acetiltransferasas/genética , Proteínas de la Membrana/genética , Mutación , Proteínas Nucleares/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcripción Genética , Activación Transcripcional , UbiquitinaciónRESUMEN
Pharmacological inhibition of mitochondrial fatty acid oxidation (FAO) has been clinically used to alleviate certain metabolic diseases by remodeling cellular metabolism. However, mitochondrial FAO inhibition also leads to mechanistic target of rapamycin complex 1 (mTORC1) activation-related protein synthesis and tissue hypertrophy, but the mechanism remains unclear. Here, by using a mitochondrial FAO inhibitor (mildronate or etomoxir) or knocking out carnitine palmitoyltransferase-1, we revealed that mitochondrial FAO inhibition activated the mTORC1 pathway through general control nondepressible 5-dependent Raptor acetylation. Mitochondrial FAO inhibition significantly promoted glucose catabolism and increased intracellular acetyl-CoA levels. In response to the increased intracellular acetyl-CoA, acetyltransferase general control nondepressible 5 activated mTORC1 by catalyzing Raptor acetylation through direct interaction. Further investigation also screened Raptor deacetylase histone deacetylase class II and identified histone deacetylase 7 as a potential regulator of Raptor. These results provide a possible mechanistic explanation for the mTORC1 activation after mitochondrial FAO inhibition and also bring light to reveal the roles of nutrient metabolic remodeling in regulating protein acetylation by affecting acetyl-CoA production.
RESUMEN
BACKGROUND: Sorafenib resistance is becoming increasingly common and disadvantageous for hepatocellular carcinoma (HCC) treatment. Ferroptosis is an iron dependent programmed cell death underlying the mechanism of sorafenib. Iron is crucial for synthesis of cofactors essential to mitochondrial enzymes and necessary for HCC proliferation, while mitochondrial iron overload and oxidative stress are associated with sorafenib induced ferroptosis. However, the crosstalk among iron homeostasis and sorafenib resistance is unclear. METHODS: We conducted bioinformatics analysis of sorafenib treated HCC datasets to analyze GCN5L1 and iron related gene expression with sorafenib resistance. GCN5L1 deleted HCC cell lines were generated by CRISPR technology. Sorafenib resistant HCC cell line was established to validate dataset analysis and evaluate the effect of potential target. RESULTS: We identified GCN5L1, a regulator of mitochondrial acetylation, as a modulator in sorafenib-induced ferroptosis via affecting mitochondrial iron homeostasis. GCN5L1 deficiency significantly increased sorafenib sensitivity in HCC cells by down-regulating mitochondrial iron transporters CISD1 expression to induce iron accumulation. Mitochondrial iron accumulation leads to an acceleration in cellular and lipid ROS. Sorafenib resistance is related to CISD1 overexpression to release mitochondrial iron and maintaining mitochondrial homeostasis. We combined CISD1 inhibitor NL-1 with sorafenib, which significantly enhanced sorafenib-induced ferroptosis by promoting mitochondrial iron accumulation and lipid peroxidation. The combination of NL-1 with sorafenib enhanced sorafenib efficacy in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that GCN5L1/CISD1 axis is crucial for sorafenib resistance and would be a potential therapeutic strategy for sorafenib resistant HCC.
Asunto(s)
Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Ferroptosis , Homeostasis , Hierro , Neoplasias Hepáticas , Mitocondrias , Sorafenib , Sorafenib/farmacología , Sorafenib/uso terapéutico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Hierro/metabolismo , Humanos , Homeostasis/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Línea Celular Tumoral , Animales , Ferroptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Ratones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: Iron overload, oxidative stress and ferroptosis are associated with liver injury in alcohol-associated liver disease (ALD), however, the crosstalk among these regulatory pathways in ALD development is unclear. METHODS: ALD mouse model and general control of amino acid synthesis 5 like 1 (GCN5L1) liver knockout mice were generated to investigate the role of GCN5L1 in ALD development. Proteomic screening tests were performed to identify the key factors mediating GCN5L1 loss-induced ALD. RESULTS: Gene Expression Omnibus data set analysis indicates that GCN5L1 expression is negatively associated with ALD progression. GCN5L1 hepatic knockout mice develop severe liver injury and lipid accumulation when fed an alcohol diet. Screening tests identified that GCN5L1 targeted the mitochondrial iron transporter CISD1 to regulate mitochondrial iron homeostasis in ethanol-induced ferroptosis. GCN5L1-modulated CISD1 acetylation and activity were crucial for iron accumulation and ferroptosis in response to alcohol exposure. CONCLUSION: Pharmaceutical modulation of CISD1 activity is critical for cellular iron homeostasis and ethanol-induced ferroptosis. The GCN5L1/CISD1 axis is crucial for oxidative stress and ethanol-induced ferroptosis in ALD and is a promising avenue for novel therapeutic strategies.
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Modelos Animales de Enfermedad , Ferroptosis , Hepatopatías Alcohólicas , Ratones Noqueados , Estrés Oxidativo , Animales , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/genética , Hepatopatías Alcohólicas/patología , Ratones , Hierro/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Etanol , Ratones Endogámicos C57BL , Humanos , Proteínas del Tejido Nervioso , Proteínas MitocondrialesRESUMEN
BACKGROUND: Inflammation resolution and cardiac repair initiation after myocardial infarction (MI) require timely activation of reparative signals. Histone lactylation confers macrophage homeostatic gene expression signatures via transcriptional regulation. However, the role of histone lactylation in the repair response post-MI remains unclear. We aimed to investigate whether histone lactylation induces reparative gene expression in monocytes early and remotely post-MI. METHODS: Single-cell transcriptome data indicated that reparative genes were activated early and remotely in bone marrow and circulating monocytes before cardiac recruitment. Western blotting and immunofluorescence staining revealed increases in histone lactylation levels, including the previously identified histone H3K18 lactylation in monocyte-macrophages early post-MI. Through joint CUT&Tag and RNA-sequencing analyses, we identified Lrg1, Vegf-a, and IL-10 as histone H3K18 lactylation target genes. The increased modification and expression levels of these target genes post-MI were verified by chromatin immunoprecipitation-qPCR and reverse transcription-qPCR. RESULTS: We demonstrated that histone lactylation regulates the anti-inflammatory and pro-angiogenic dual activities of monocyte-macrophages by facilitating reparative gene transcription and confirmed that histone lactylation favors a reparative environment and improves cardiac function post-MI. Furthermore, we explored the potential positive role of monocyte histone lactylation in reperfused MI. Mechanistically, we provided new evidence that monocytes undergo metabolic reprogramming in the early stage of MI and demonstrated that dysregulated glycolysis and MCT1 (monocarboxylate transporter 1)-mediated lactate transport promote histone lactylation. Finally, we revealed the catalytic effect of IL (interleukin)-1ß-dependent GCN5 (general control non-depressible 5) recruitment on histone H3K18 lactylation and elucidated its potential role as an upstream regulatory element in the regulation of monocyte histone lactylation and downstream reparative gene expression post-MI. CONCLUSIONS: Histone lactylation promotes early remote activation of the reparative transcriptional response in monocytes, which is essential for the establishment of immune homeostasis and timely activation of the cardiac repair process post-MI.
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Histonas , Infarto del Miocardio , Humanos , Histonas/metabolismo , Activación Transcripcional , Infarto del Miocardio/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismoRESUMEN
In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a â¼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.
RESUMEN
Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Giberelinas , Histona Acetiltransferasas , Transducción de Señal , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Giberelinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Acetilación , Flores/crecimiento & desarrollo , Flores/genética , Flores/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Histonas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genéticaRESUMEN
Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Here, using Drosophila, we perform label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. We further characterize the dynamics of protein acetylation with age, which exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, we reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, our study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.
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Proteínas de Drosophila , Histona Acetiltransferasas , Lisina Acetiltransferasas , Animales , Acetilación , Drosophila , Histona Acetiltransferasas/metabolismo , Lisina/metabolismo , Lisina Acetiltransferasas/metabolismo , Proteínas de Drosophila/metabolismoRESUMEN
The Notch signaling pathway is related to endothelial dysfunction in coronary atherosclerosis. Our objective was to explore the role of Notch signaling in coronary microvascular dysfunction (CMD). CMD models were constructed by sodium laurate injection in vivo and homocysteine (Hcy) stimulation in vitro. The binding ability of Notch Intracellular Domain (NICD)/H3K9Ac/GCN5 (General Control Non-derepressible 5) to Neuregulin-1 (Nrg-1) promoter was examined by chromatin immunoprecipitation. Immunofluorescence staining was conducted to detect CD31 positive cells, NICD localization, and co-localization of NICD and GCN5. Flow cytometry and Tunel staining were conducted to identify the apoptosis. Hematoxylin and eosin staining, quantitative real-time PCR, western blot, immunohistochemical staining, co-immunoprecipitation, and double luciferase report analysis were also conducted. Notch signaling pathway-related protein levels were decreased, levels of Nrg-1 and the phosphorylation of ErbB2 and ErbB4 were enhanced in CMD models. Interference with Nrg-1 further increased the apoptosis in Hcy-induced cardiac microvascular endothelial cells (CMECs). Meanwhile, the activation of the Notch signaling pathway increased the levels of Nrg-1 and the phosphorylation of ErbB2 and ErbB4, as well as inhibited the apoptosis induced by Hcy. Furthermore, NICD and histone acetyltransferase enzyme GCN5 could regulate Nrg-1 promoter activity by affecting the expression of acetylation-modified protein H3K9Ac. In addition, NICD also interacted with GCN5. In vivo results also confirmed that the activation of the Notch signal alleviated CMD. Notch signaling pathway regulates Nrg-1 level through synergistic interaction with GCN5, thereby mitigating CMD.
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Células Endoteliales , Isquemia Miocárdica , Humanos , Células Endoteliales/metabolismo , Neurregulina-1/metabolismo , Neurregulina-1/farmacología , Código de Histonas , Apoptosis , Transducción de Señal , Receptor ErbB-4/metabolismo , Isquemia Miocárdica/metabolismo , Receptor Notch1RESUMEN
Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.
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Antimaláricos , Artemisininas , Malaria Falciparum , Humanos , Plasmodium falciparum/metabolismo , Artemisininas/farmacología , Artemisininas/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Glucosa/metabolismo , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Resistencia a Medicamentos/genéticaRESUMEN
Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Cinetocoros/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteína A Centromérica/química , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/química , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Mutación Missense , Organismos Modificados Genéticamente , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In the process of orthodontic tooth movement (OTM), periodontal ligament fibroblasts (PDLFs) must undergo osteogenic differentiation. OTM increased the expression of Zinc finger and BTB domain-containing 16 (ZBTB16), which is implicated in osteogenic differentiation. Our goal was to investigate the mechanism of PDLF osteogenic differentiation mediated by ZBTB16. The OTM rat model was established, and PDLFs were isolated and exposed to mechanical force. Hematoxylin-eosin staining, Alizarin Red staining, immunofluorescence, and immunohistochemistry were carried out. The alkaline phosphatase (ALP) activity was measured. Dual-luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted. In OTM models, ZBTB16 was significantly expressed. Additionally, there was an uneven distribution of PDLFs in the OTM group, as well as an increase in fibroblasts and inflammatory infiltration. ZBTB16 interference hindered PDLF osteogenic differentiation and decreased Wnt and ß-catenin levels. Meanwhile, ZBTB16 activated the Wnt/ß-catenin pathway. ZBTB16 also enhanced the expression of the osteogenic molecules osterix, osteocalcin (OCN), osteopontin (OPN), and bone sialo protein (BSP) at mRNA and protein levels. The interactions between Wnt1 and ZBTB16, as well as GCN5 and ZBTB16, were also verified. The adeno-associated virus-shZBTB16 injection also proved to inhibit osteogenic differentiation and reduce tooth movement distance in in vivo tests. ZBTB16 was up-regulated in OTM. Through acetylation modification of ZBTB16, GCN5 regulated the Wnt/ß-catenin signaling pathway and further mediated PDLF osteogenic differentiation.
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Osteogénesis , beta Catenina , Ratas , Animales , Osteogénesis/genética , beta Catenina/metabolismo , Acetilación , Técnicas de Movimiento Dental , Ligamento Periodontal , Vía de Señalización Wnt/genética , Diferenciación Celular , Células Cultivadas , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Histona Acetiltransferasas/metabolismoRESUMEN
Acetylation is a conserved modification catalyzed by acetyltransferases that play prominent roles in a large number of biological processes. Members of the general control non-repressible 5 (GCN5)-N-acetyltransferase (GNAT) protein superfamily are widespread in all kingdoms of life and are characterized by highly conserved catalytic fold, and can acetylate a wide range of substrates. Although the structures and functions of numerous eukaryotic GNATs have been identified thus far, many GNATs in microorganisms remain structurally and functionally undescribed. Here, we determined the crystal structure of the putative GCN5-N-acetyltransferase PgbP in complex with CoA in Serratia marcescens FS14. Structural analysis revealed that the PgbP dimer has two cavities, each of which binds a CoA molecule via conserved motifs of the GNAT family. In addition, the biochemical studies showed that PgbP is a prodigiosin-binding protein with high thermal stability. To our knowledge, this is the first view of GNAT binding to secondary metabolites and it is also the first report of prodigiosin binding protein. Molecular docking and mutation experiments indicated that prodigiosin binds to the substrate binding site of PgbP. The structure-function analyses presented here broaden our understanding of the multifunctionality of GNAT family members and may infer the mechanism of the multiple biological activities of prodigiosin.
Asunto(s)
Prodigiosina , Serratia marcescens , Serratia marcescens/metabolismo , Proteínas Portadoras/metabolismo , Secuencia de Aminoácidos , Simulación del Acoplamiento Molecular , Acetiltransferasas/metabolismoRESUMEN
Interleukin-17 (IL-17), a potent proinflammatory cytokine, can trigger the metastasis of non-small cell lung cancer (NSCLC). However, the underlying mechanism involved in IL-17-induced NSCLC cell metastasis remains unclear. In this study, we found that not only the expression of IL-17, IL-17RA, and/or general control nonrepressed protein 5 (GCN5), SRY-related HMG-BOX gene 4 (SOX4), and matrix metalloproteinase 9 (MMP9) was increased in the NSCLC tissues and in the IL-17-stimulated NSCLC cells, but also IL-17 treatment could enhance NSCLC cell migration and invasion. Further mechanism exploration revealed that IL-17-upregulated GCN5 and SOX4 could bind to the same region (-915 to -712 nt) of downstream MMP9 gene promoter driving its gene transcription. In the process, GCN5 could mediate SOX4 acetylation at lysine 118 (K118, a newly identified site) boosting MMP9 gene expression as well as cell migration and invasion. Moreover, the SOX4 acetylation or MMP9 induction and metastatic nodule number in the lung tissues of the BALB/c nude mice inoculated with the NSCLC cells stably infected by corresponding LV-shGCN5 or LV-shSOX4, LV-shMMP9 plus IL-17 incubation were markedly reduced. Overall, our findings implicate that NSCLC metastasis is closely associated with IL-17-GCN5-SOX4-MMP9 axis.