RESUMEN
BACKGROUND: Klebsiella pneumoniae is widely distributed in water and plays a major role in both human and poultry infections. Many K. pneumoniae strains form biofilms on various surfaces, enhancing their pathogenicity and resistance to antibiotics. The water supply pipeline of chicken farms has become a hotbed for the growth of K pneumoniae biofilm because of its humid environment, and because the chicken drinking water pipeline is thin, it is easily blocked by the biofilm, and the diffused cells can cause repeated and persistent infections. Iron is vital to the growth of microorganisms and the formation of biofilms. Therefore, the aim of this study was to examine the effects of iron on K. pneumoniae biofilm formation and any associated metabolic changes to provide a rationale for reducing the formation of biofilms. RESULTS: Biofilm formation was enhanced to the greatest extent by the presence of 0.16 mM FeCl2, producing a denser structure under electron microscopy. The number of biofilm-forming and planktonic bacteria did not change, but protein and polysaccharide concentrations in the bacterial extracellular polymeric substances (EPS) were significantly increased by iron supplementation. To clarify this mechanism, intracellular metabolomic analysis was carried out, showing that the differential, down-regulated metabolites included succinic acid. The addition of 1.7 mM succinic acid counteracted the biofilm-forming effect of iron, with no bactericidal side effects. CONCLUSION: This study demonstrates the importance of succinic acid and iron in K. pneumoniae biofilms, and provides insight into the formation of K. pneumoniae biofilms and direction for the development of new antibacterial agents.
Asunto(s)
Klebsiella pneumoniae , Ácido Succínico , Antibacterianos/farmacología , Biopelículas , Hierro/farmacología , Ácido Succínico/farmacologíaRESUMEN
Osteoporotic fractures cause major morbidity and mortality in the aging population. Genome-wide association studies (GWAS) have identified USF3 as the novel susceptibility gene of osteoporosis. However, the functional role in bone metabolism and the target gene of the basic helix-loop-helix transcription factor USF3 are unclear. Here, we show that USF3 enhances osteoblast differentiation and suppresses osteoclastogenesis in cultured human osteoblast-like U-2OS cells. Mechanistic studies revealed that transcription factor USF3 antagonistically interacts with anti-osteogenic TWIST1/TCF12 heterodimer in the WNT16 and RUNX2 promoter, and counteracts CREB1 and JUN/FOS in the RANKL promoter. Importantly, the osteoporosis GWAS variant g.1744A>G (rs2908007A>G) located in the WNT16 promoter confers G-allele-specific transcriptional modulation by USF3, TWIST1/TCF12 and TBX5/TBX15, and USF3 transactivates the osteoclastogenesis suppressor WNT16 promoter activity and antagonizes the repression of WNT16 by TWIST1 and TCF12. The risk G allele of osteoporosis GWAS variant g.3260A>G (rs4531631A>G) in the RANKL promoter facilitates the binding of CREB1 and JUN/FOS and enhances transactivation of the osteoclastogenesis contributor RANKL that is inhibited by USF3. Our findings uncovered the functional mechanisms of osteoporosis novel GWAS-associated gene USF3 and lead single nucleotide polymorphisms rs2908007 and rs4531631 in the regulation of bone formation and resorption.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Estudio de Asociación del Genoma Completo , Osteoporosis , Anciano , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Humanos , Osteoblastos , Osteoporosis/genética , Polimorfismo de Nucleótido Simple , Ligando RANK/genética , Proteínas de Dominio T Box/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Upstream transcription factor family member 3 (USF3) c.3781C>A (rs1026364) in the 3'-untranslated region (3'-UTR) has been firmly associated with bone mineral density (BMD) in genome-wide association study (GWAS). However, the molecular mechanism by which it influences BMD and osteoporosis is unknown. Bioinformatics analyses suggested that the risk c.3781A allele creates a target site for hsa-miR-345-5p binding. Luciferase assay validated that the c.3781A allele displayed significantly lower luciferase activities than the c.3781C allele in the human osteoblast cell line hFOB1.19, osteosarcoma cell lines U-2OS and Saos-2, and embryonic kidney cell line 293T. Furthermore, hsa-miR-345-5p regulated USF3 expression on both messenger RNA and protein levels in hFOB1.19 and U937 cells with heterozygous A/C genotype. Transfection of hsa-miR-345-5p antagomiR in heterozygous hFOB1.19 cells significantly increased the expression of osteogenic marker genes RUNX2, OSTERIX, COL1A1, ALP, OPN, OCN, and alkaline phosphatase activity and matrix mineralization level. Importantly, we found that hsa-miR-345-5p also inhibits osteoblast maturation in cell lines U-2OS with hsa-miR-345-5p nonbinding C/C genotype by targeting RUNX3 and SMAD1. Our findings uncovered a novel pathogenetic mechanism of osteoporosis by GWAS variant c.3781C>A-mediated disruption of hsa-miR-345-5p binding at the 3'-UTR of USF3 and the functional role of hsa-miR-345-5p in osteogenic differentiation.
Asunto(s)
Alelos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , MicroARNs/genética , Osteoporosis/diagnóstico , Osteoporosis/genética , Regiones no Traducidas 3' , Diferenciación Celular/genética , Línea Celular , Biología Computacional/métodos , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Conformación de Ácido Nucleico , Osteoblastos/citología , Osteoblastos/metabolismo , Interferencia de ARN , ARN Mensajero/genética , TranscriptomaRESUMEN
Previous genome-wide linkage and association studies have identified an osteoporosis-associated locus at 1p36 that harbors SNPs rs34920465 and rs6426749. The 1p36 locus also comprises the WNT4 gene with known role in bone metabolism and functionally unknown ZBTB40/lncRNA ZBTB40-IT1 genes. How these might interact to contribute to osteoporosis susceptibility is not known. In this study, we show that lncRNA ZBTB40-IT1 is able to suppress osteogenesis and promote osteoclastogenesis by regulating the expression of WNT4, RUNX2, OSX, ALP, COL1A1, OPG and RANKL in U-2OS and hFOB1.19 cell lines, whereas ZBTB40 plays an opposite role in bone metabolism. Treatment with parathyroid hormone significantly downregulates the expression of ZBTB40-IT1 in U-2OS cell lines. ZBTB40 can suppress ZBTB40-IT1 expression but has no response to parathyroid hormone treatment. Dual-luciferase assay and biotin pull-down assay demonstrate that osteoporosis GWAS lead SNPs rs34920465-G and rs6426749-C alleles can respectively bind transcription factors JUN::FOS and CREB1, and upregulate ZBTB40 and ZBTB40-IT1 expression. Our study discovers the critical role of ZBTB40 and lncRNA ZBTB40-IT1 in bone metabolism, and provides a mechanistic basis for osteoporosis GWAS lead SNPs rs34920465 and rs6426749.
Asunto(s)
Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Osteogénesis/genética , Osteoporosis , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante , Alelos , Línea Celular Tumoral , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/farmacología , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genéticaRESUMEN
We recently reported that the expression of the synaptic form of acetylcholinesterase (AChE) is induced during apoptosis in various cell types in vitro. Here, we provide evidence to confirm that AChE is expressed during ischemia-reperfusion (I/R)-induced apoptosis in vivo. Renal I/R is a major cause of acute renal failure (ARF), resulting in injury and the eventual death of renal cells due to a combination of apoptosis and necrosis. Using AChE-deficient mice and AChE inhibitors, we investigated whether AChE deficiency or inhibition can protect against apoptosis caused by I/R in a murine kidney model. Unilateral clamping of renal pedicles for 90 min followed by reperfusion for 24 h caused significant renal dysfunction and injury. Both genetic AChE deficiency and chemical inhibition of AChE (provided by huperzine A, tacrine and donepezil) significantly reduced the biochemical and histological evidence of renal dysfunction following I/R. Activation of caspases-8, -9, -12, and -3 in vivo were prevented and associated with reduced levels of cell apoptosis and cell death. A further investigation also confirmed that AChE deficiency down-regulated p53 induction and phosphorylation at serine-15, and decreased the Bax/Bcl-2 ratio during I/R. In conclusion, our study demonstrates that AChE may be a pro-apoptotic factor and the inhibition of AChE reduces renal I/R injury. These findings suggest that AChE inhibitors may represent a therapeutic strategy for protection against ischemic acute renal failure.
Asunto(s)
Acetilcolinesterasa/deficiencia , Apoptosis , Pruebas de Función Renal , Riñón/fisiopatología , Daño por Reperfusión/enzimología , Daño por Reperfusión/fisiopatología , Proteína p53 Supresora de Tumor/metabolismo , Acetilcolinesterasa/biosíntesis , Animales , Caspasas/metabolismo , Creatinina/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Activación Enzimática , Inducción Enzimática , Riñón/enzimología , Riñón/patología , Túbulos Renales/patología , Túbulos Renales/fisiopatología , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Daño por Reperfusión/patología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Acetylcholinesterase (AChE) expression may be induced during apoptosis in various cell types. Here, we used the C-terminal of AChE to screen the human fetal brain library and found that it interacted with Ran-binding protein in the microtubule-organizing center (RanBPM). This interaction was further confirmed by coimmunoprecipitation analysis. In HEK293T cells, RanBPM and AChE were heterogeneously expressed in the cisplatin-untreated cytoplasmic extracts and in the cisplatin-treated cytoplasmic or nuclear extracts. Our previous studies performed using morphologic methods have shown that AChE translocates from the cytoplasm to the nucleus during apoptosis. Taken together, these results suggest that RanBPM is an AChE-interacting protein that is translocated from the cytoplasm into the nucleus during apoptosis, similar to the translocation observed in case of AChE.
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Acetilcolinesterasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Riñón/metabolismo , Proteínas Nucleares/metabolismo , Sitios de Unión , Línea Celular , Humanos , Unión Proteica , Transporte de Proteínas/fisiologíaRESUMEN
Bcl6B, also known as BAZF, plays important roles in the immune response, repression of cancers, and maintenance of spermatogonial stem cells in mammals. In this study, the homologous gene bcl6b and its 5 alternative splicing variants, namely bcl6bX1 to bcl6bX5, were isolated from medaka fish, Oryzias latipes. Medaka bcl6b possesses conserved domains such as BTB domain, RD2 domain and four zinc fingers. Medaka bcl6bX1 to bcl6bX3 possess all three previously mentioned domains with minor differences in sequences. Medaka bcl6bX4 possesses only the BTB domain due to premature stopping, and bcl6bX5 possesses both the BTB domain and zinc fingers without the RD2 domain. Medaka bcl6b was expressed in the tissues including the brain, heart, gill, muscle, spleen, kidney, intestine, ovary and testes of adult fish. Medaka bcl6b was expressed in the embryos from very early stage, and could be detected clearly in the developing eyes by RT-PCR and in situ hybridization. Medaka bcl6b could respond to the stimuli of polyI:C and LPS in the kidney and spleen. Medaka bcl6bX1 to bcl6bX3 were the majority of the variants expressed in the adult tissues and the embryos, and were the major response to the stimulation of polyI:C and LPS in the spleen. These results suggested that bcl6b, including its isoforms, could function in various tissues and embryogenesis. Moreover, bcl6b might be a factor for immune response in medaka.
Asunto(s)
Empalme Alternativo , Desarrollo Embrionario , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Oryzias/fisiología , Proteínas Represoras/metabolismo , Empalme Alternativo/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Embrión no Mamífero/inmunología , Embrión no Mamífero/fisiología , Ojo/embriología , Ojo/metabolismo , Proteínas de Peces/química , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inductores de Interferón/farmacología , Riñón/efectos de los fármacos , Riñón/crecimiento & desarrollo , Riñón/inmunología , Riñón/metabolismo , Lipopolisacáridos/farmacología , Especificidad de Órganos , Oryzias/embriología , Oryzias/crecimiento & desarrollo , Oryzias/inmunología , Poli I-C/farmacología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución Aleatoria , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Bazo/efectos de los fármacos , Bazo/crecimiento & desarrollo , Bazo/inmunología , Bazo/metabolismoRESUMEN
The classical function of acetylcholinesterase (AChE) is to terminate synaptic transmission at cholinergic synapses by rapidly hydrolyzing the neurotransmitter acetylcholine (ACh). Non-classical functions of AChE involve accelerating the assembly of Abeta peptide into amyloid fibrils and participating in haematopoiesis and neurite growth. Although numerous antibodies have been raised against AChE, many researchers have questioned their reliability to identify the AChE in situ, especially with the regard to its non-classical roles. Researchers attended the Ninth International Meeting on Cholinesterase raised this question by showing different Western blot patterns of AChE detected by different Abs. Producing more effective and reliable Abs for measuring AChE in vivo or in situ has become an important issue in many scientific fields. In this paper, we introduce a monoclonal antibody raised against synaptic AChE that we identified by Western blot assays, immunofluorescent staining and immunoprecipitation of AChE, and mass spectrometry. Our results strongly demonstrate the specificity of our monoclonal antibody to recognize synaptic AChE; hence our antibody can be used as an effective tool to study the various functions of AChE. Since the apoptosis-related AChE was its synaptic form, our antibody can be used as a tool to detect apoptotic cells.
Asunto(s)
Acetilcolinesterasa/inmunología , Anticuerpos Monoclonales/inmunología , Apoptosis , Sinapsis/enzimología , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The SOST gene encodes sclerostin, a C-terminal cysteine knot-like domain containing key negative regulator of osteoblastic bone formation that inhibits LRP5/6-mediated canonical Wnt signaling. Numerous single nucleotide polymorphisms (SNPs) in the SOST locus are firmly associated with bone mineral density (BMD) and fracture in genome-wide association studies (GWAS) and candidate gene association studies. However, the validation and mechanistic elucidation of causal genetic variants, especially for SNPs located beyond the promoter-proximal region, remain largely unresolved. By employing computational and experimental approaches, here we identify four SNPs rs1230399, rs7220711, rs1107748 and rs75901553 as functional variants which display allelic variation in SOST gene expression. The osteoporosis associated SNP rs1230399 in the SOST distal upstream regulatory region shows FOXA1 binding activity with subsequent transinactivation in a T allele-specific manner. The BMD GWAS lead SNPs rs7220711 and rs1107748 both reside in the 52-kb regulatory element deletion 35-kb downstream of the SOST gene which leads to Van Buchem disease. The rs7220711-A has a higher affinity for the transcriptional repressors MAFF or MAFK homodimers than rs7220711-G, while rs1107748 confers C allele specific transcriptional enhancer activity via a CTCF binding element. The variant rs75901553 C>T located in a conserved site of the SOST 3' UTR abolishes a target binding site for miR-98-5p which is negatively responsive to parathyroid hormone or 17ß-estradiol in osteoblastic cell lines. Our findings uncover the biological consequences of four independent genetic variants in the SOST region and their important roles in SOST expression via diverse mechanisms, providing new insights into the genetics and molecular pathogenesis of osteoporosis.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Biología Computacional/métodos , Sitios Genéticos , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad , Osteoporosis/genética , Polimorfismo de Nucleótido Simple/genética , Regiones no Traducidas 3'/genética , Proteínas Adaptadoras Transductoras de Señales , Alelos , Secuencia de Bases , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Elementos de Facilitación Genéticos/genética , Frecuencia de los Genes/genética , Células HEK293 , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Hormona Paratiroidea/farmacología , Unión Proteica/efectos de los fármacos , Multimerización de ProteínaRESUMEN
Fundc1 is a mitochondrial outer membrane protein and plays important roles in mitochondria fission and hypoxia-induced mitophagy in mammalian cells. However, there is no relevant report of fundc1 in fish. In the present study, we cloned a 942bp fundc1 cDNA from rare minnow. The cDNA, designated as Grfundc1 cDNA, contains an open reading frame (ORF) of 459bp which encodes a polypeptide of 152 amino acid residues. Comparisons of deduced amino acid sequences demonstrated that Grfundc1 was highly homologous with those of other vertebrates. RT-PCR and real time PCR detection revealed that the transcripts of Grfundc1 were not detectable in the unfertilized eggs and had high levels at blastula and gastrula stages. Whole mount in situ hybridization analysis observed that Grfundc1 was ubiquitously expressed at early stage and later riched in specific regions, such as brain, branchial arch, eye and somite during embryogenesis. Grfundc1 was expressed in all the tissues of rare minnow adult, including brain, liver, gill, eyes, heart, kidney, intestine, muscle, testis and ovary. The expression of Grfundc1 in the brain, gill, heart and eye of rare minnow adult was significantly down-regulated by hypoxia. Similar hypoxic response was observed in the rare minnow embryos at 48hpf following hypoxia exposure. Functional analysis showed that knockdown of Grfundc1 significantly caused defects in the body axis and dorsal neural tissues of rare minnow embryos. These results indicate that Grfundc1 may play important roles in embryogenesis in fish.
Asunto(s)
Cyprinidae/genética , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Animales , Cyprinidae/embriología , Proteínas de Peces/metabolismo , Hipoxia/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Especificidad de Órganos , Estrés FisiológicoRESUMEN
OBJECTIVES: Genome-wide association studies (GWASs) have revealed many SNPs and genes associated with osteoporosis. However, influence of these SNPs and genes on the predisposition to osteoporosis is not fully understood. We aimed to identify osteoporosis GWASs-associated SNPs potentially influencing the binding affinity of transcription factors and miRNAs, and reveal enrichment signaling pathway and "hub" genes of osteoporosis GWAS-associated genes. METHODS: We conducted multiple computational analyses to explore function and mechanisms of osteoporosis GWAS-associated SNPs and genes, including SNP conservation analysis and functional annotation (influence of SNPs on transcription factors and miRNA binding), gene ontology analysis, pathway analysis and protein-protein interaction analysis. RESULTS: Our results suggested that a number of SNPs potentially influence the binding affinity of transcription factors (NFATC2, MEF2C, SOX9, RUNX2, ESR2, FOXA1 and STAT3) and miRNAs. Osteoporosis GWASs-associated genes showed enrichment of Wnt signaling pathway, basal cell carcinoma and Hedgehog signaling pathway. Highly interconnected "hub" genes revealed by interaction network analysis are RUNX2, SP7, TNFRSF11B, LRP5, DKK1, ESR1 and SOST. CONCLUSIONS: Our results provided the targets for further experimental assessment and further insight on osteoporosis pathophysiology.
Asunto(s)
Predisposición Genética a la Enfermedad , MicroARNs/genética , Osteoporosis/genética , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Estudio de Asociación del Genoma Completo , Genotipo , HumanosRESUMEN
RNA modification, involving in a wide variety of cellular processes, has been identified over 100 types since 1950s. N(6)-methyladenosine (m6A), as one of the most abundant RNA modifications, is found in several RNA species and predominantly located in the stop codons, long internal exons as well as 3'UTR. It was reported that m6A modification preferentially appears after G in the conserved motif RRm6ACH (R = A/G and H = A/C/U). There are two families of enzymes responsible for maintaining the balance of m6A modification: m6A methyltransferases and demethylases, which add and remove methyl marks for adenosine of RNA, respectively. METTL3 complex, the m6A methyltransferases, and two kinds of demethylases including Fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5) are characterized thus far. Besides the "writers" and "erasers", m6A specific recognizing proteins, such as the YTH (YT521-B homology) domain family proteins, also have attracted significant attention. Herein, we focus on the recent progress in understanding the biological/biochemical functions and structures of proteins responsible for the m6A modification and recognition. Detailed analyses of these important proteins are essential for the further study of their biological function and will also guide us in designing more potent and specific small-molecule chemical inhibitors for these targets.
Asunto(s)
Adenosina/análogos & derivados , Proteínas/metabolismo , Adenosina/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Proteínas/químicaRESUMEN
Apoptosis, also known as programmed cell death, plays an essential role in eliminating excessive, damaged or harmful cells. Previous work has demonstrated that anticancer drugs induce cell apoptosis by inducing cytotoxicity. In recent years, several reports demonstrated modulated expression of DNA methyltransferases 1 (DNMT1) and acetylcholinesterase (AChE) in a variety of tumors. In this study, we showed that the expression of DNMT1 was decreased and the methylation of CpGs in the promoter of AChE was reduced in anticancer drugs-induced apoptotic hepatocellular carcinoma cells. Silencing of DNMT1 expression by AZA or RNA interference (RNAi) restored AChE production and inhibition of AChE expression by RNAi protected HCC cells from anticancer drugs-induced apoptosis. Furthermore, we demonstrated that the regulation of AChE by DNMT1 was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis. In addition, immunohistochemical staining showed that P-p38, DNMT1 and AChE were aberrantly expressed in a subset of HCC tumors. Taken together, we demonstrated the regulation of AChE by DNMT1 and further, we found that this regulation was involved in the phosphorylated p38 pathway in anticancer drugs-induced apoptosis.
Asunto(s)
Acetilcolinesterasa/genética , Antineoplásicos/farmacología , Carcinoma Hepatocelular/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Acetilcolinesterasa/metabolismo , Apoptosis/efectos de los fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Cisplatino/farmacología , Islas de CpG , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Decitabina , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
More than 100 types of RNA modifications have been identified so far, which are involved in a variety of cellular processes. N6-methyladenosine (m6A), as one most abundant RNA modification, is found in several RNA species, and mainly located in the stop codons, long internal exons as well as 3'UTR. It was reported that m6A modification is preferred after G in the conserved sequence RRm6ACH (R = A/G and H = A/C/U). There are two families of enzymes responsible for maintaining the balance of m6A methylation: RNA methyltransferases and demethylases, which add and remove methyl marks from RNA, respectively. METTL3 complex, the m6A RNA methyltransferase, has been identified, and two kinds of demethylases are characterized thus far, including Fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5). Besides the "writers" and "erasers" for m6A, m6A specific recognizing protein, such as the YTH domain, also has attracted significant attention. Herein, we will focus on the recent progress in understanding biological/biochemical functions and structures of proteins responsible for the m6A RNA modification and recognition. Detailed analysis of these important proteins will guide us in designing target-specific small molecule chemical probes and inhibitors.
RESUMEN
In recent years, widespread antisense transcripts have been identified systematically in mammalian cells and are known to regulate gene expression, although their functional significance remains largely unknown. Previous work has identified that acetylcholinesterase (AChE) is expressed aberrantly in various malignant tumors and function as a tumor growth suppressor. However, the mechanism of AChE gene regulation in tumors remains unclear. In this study, we show that the AChE antisense RNA (AChE-AS) play an important role in AChE expression regulation. An inverse relationship was identified between AChE-AS and AChE expression in hepatocellular carcinoma and hepatoma cells. The silenced AChE-AS corresponds to elevated expression of AChE. Furthermore, we demonstrated that reduced AChE-AS increased H3K4 methylation and decreased H3K9 methylation in the AChE promoter region. As expected, elevated AChE levels induced by inhibition of AChE-AS enhanced anticarcinogen-induced apoptosis. These observations demonstrated that AChE-AS modulates AChE expression and exerts an anti-apoptotic effect through direct repression of AChE expression in HCC cells. Thus, natural antisense RNA may play an important role in AChE regulation via affecting the epigenetic modification in the AChE promoter region.
Asunto(s)
Acetilcolinesterasa/genética , Carcinoma Hepatocelular/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , ARN sin Sentido/genética , Acetilcolinesterasa/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Cisplatino/farmacología , Metilación de ADN , Células Hep G2 , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Lisina/metabolismo , Metilación , Mitomicina/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MiR-132 is enriched in the central nerve system and is thought to be involved in neuronal development, maturation and function, and to be associated with several neurological disorders including Alzheimer's disease. In addition to its documented neuronal functions, an emerging role for miR-132 in tumorigenesis has been suggested. Recently, hsa-miR-132 was shown to be modulated in different tumor types. However, its role in non-small cell lung cancer (NSCLC) remains unclear. Here, we show that hsa-miR-132 can initiate apoptosis in NSCLC cells to dramatically attenuate tumor formation in nude mice independent of its effect on the proliferation/apoptosis-associated gene, acetylcholinesterase (AChE). Interestingly, hsa-miR-132 has no pro-apoptotic effect in normal pulmonary trachea epithelium. Taken together, these results suggest that hsa-miR-132 represses NSCLC growth by inducing apoptosis independent of AChE.
Asunto(s)
Acetilcolinesterasa/metabolismo , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , MicroARNs/metabolismo , Animales , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genéticaRESUMEN
The apoptosis pathway has been proposed to be involved in causing neuronal cell death in the pathogenesis of Parkinson's disease. However, the details of this pathway are poorly understood. Previous research has shown increased acetylcholinesterase expression during apoptosis in various cell types, which suggests that acetylcholinesterase has a potential role in neuronal cell death. In this study, we found that acetylcholinesterase protein expression increased and caspase-3 was activated in PC12 cells treated with 1-methyl-4-phenylpyridinium. Furthermore, the genetic or pharmacological inhibition of acetylcholinesterase was shown to protect PC12 cells from MPP+ induced apoptotic cell death. To study the function of acetylcholinesterase as a mechanism of neuronal cell death in vivo, we subsequently established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Parkinson's disease mouse model utilizing acetylcholinesterase-deficient mice. Studies in these mice revealed reduced dopaminergic neuron loss and lower expression levels of apoptotic proteins in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated heterozygous mice compared to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated wild-type mice. We conclude that it is highly probable that acetylcholinesterase is involved in the pathogenesis of the neurotoxin model of Parkinson's disease via apoptosis. Specifically, a deficiency or inhibition of acetylcholinesterase can decrease apoptosis and protect dopaminergic neurons in the neurotoxin model of Parkinson's disease.
Asunto(s)
Acetilcolinesterasa/deficiencia , Apoptosis , Neuronas Dopaminérgicas/enzimología , Trastornos Parkinsonianos/patología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , 1-Metil-4-fenilpiridinio/farmacología , Acetilcolinesterasa/genética , Alcaloides/farmacología , Animales , Inhibidores de la Colinesterasa/farmacología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células PC12 , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/enzimología , Ratas , Sesquiterpenos/farmacología , Sustancia Negra/enzimología , Sustancia Negra/patologíaRESUMEN
Acetylcholinesterase (AChE) is emerging as an important contributor to apoptosis in various cell types. However, overexpression of AChE does not initiate apoptosis, and cells which express AChE at basal levels grow normally, suggesting that AChE may function differently between normal and apoptotic conditions. In this study, we determined that an AChE-derived protein (â¼55 kDa) positively correlated with cellular apoptotic levels. The 55 kDa AChE protein was not a result of a novel splice variant of the AChE primary transcript. Instead, it was determined to be a cleaved fragment of the full-length 68 kDa AChE protein that could not be inhibited by cycloheximide (CHX) but could be suppressed by caspase inhibitors in apoptotic PC-12 cells. Furthermore, activation of the Akt cascade abolished the 55 kDa protein, and both AChE protein forms (68 and 55 kDa) accumulated in the nucleus during apoptosis. In a mouse model for ischemia/reperfusion (I/R)-induced acute renal failure, the 55 kDa AChE protein was detected in the impaired organs but not in the normal ones, and its levels correlated with the genotype of the mice. In summary, a 55 kDa AChE protein resulting from the cleavage of 68 kDa AChE is induced during apoptosis, and it is negatively regulated by the Akt pathway. This study suggests that an alternative form of AChE may play a role in apoptosis.