RESUMEN
Acetylation of histones, the major protein component of eukaryotic chromosomes, contributes to the epigenetic regulation of gene expression and is also involved in cancer development. A recent study revealed the correlation between tumour formation and acetylation level of lysine K18 on histone H3. In this study, we developed two colorimetric in vitro assays using gold nanoparticles (AuNPs) for identification of lysine K18 acetylation on histone H3 peptide. In assay I, citrate ion-capped AuNP without further modification was employed. Simply mixing the K18 peptide with AuNP solution leads to distinct particle aggregation, relative to that by non-acetylated or lysine K14 acetylated control peptides. In assay II, an AuNP-peptide-antibody composite was synthesized and used as both the sensing probe and the transducing element. By mixing the sample peptides with the composite solution followed by PBS screening, different aggregation behaviours were observed between the K18 acetylated target peptide and the control sequences. Both assays are capable of identifying the acetylated peptides, and also differentiating the distinctive acetylation positions that differ merely by a distance of three amino acids.
Asunto(s)
Técnicas Biosensibles , Histonas/química , Lisina/química , Nanopartículas del Metal/química , Péptidos/química , Procesamiento Proteico-Postraduccional , Acetilación , Secuencia de Aminoácidos , Anticuerpos/química , Cromatina/química , Cromatina/metabolismo , Colorimetría/métodos , Colorimetría/normas , Floculación , Oro/química , Histonas/metabolismo , Lisina/metabolismo , Péptidos/síntesis química , Péptidos/metabolismoRESUMEN
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación/fisiología , Regulación hacia Arriba/fisiología , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Células HEK293 , Humanos , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteínas de Unión al ARN/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Factores de Transcripción , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitinación/efectos de los fármacos , Ubiquitinación/efectos de la radiación , Rayos Ultravioleta , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell-virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1S-specific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1S-dependent transcription through the interaction with XBP-1S.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismo , Proteínas de Unión al ADN/química , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/química , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb) and is recently identified as a novel positive regulator of p53. We previously showed the basic region (BR) of HEXIM1 mediates the binding of HEXIM1 to a nucleolar protein, nucleophosmin (NPM), and can be ubiquitinated by human double minute 2 protein. Here we identify a cytotoxic peptide derived from the BR of HEXIM1. When fused with a cell-penetrating peptide, the HEXIM1 BR peptide triggers rapid cytotoxic effect independent of p53. Similarly, when the BR peptide is linked with a breast cancer cell targeting peptide, LTV, the LTV-BR fusion peptide exhibits specific killing of breast cancer cells, which is not observed with the commonly used cytotoxic peptide, KLA. Importantly, the BR peptide fails to enter cells by itself and does not induce any cytotoxic effects when it is not guided by any cell-penetrating or cancer targeting peptides. We showed that HEXIM1 BR peptide depolarizes mitochondrial membrane potential in a p53-dependent manner and its cell-killing activity is not suppressed by caspase inhibition. Furthermore, we observed an accumulation of the internalized BR peptide in the nucleoli of treated cells and an altered localization of NPM. These results illustrate a novel mechanism which the BR peptide induces cell death and can potentially be used as a novel therapeutic strategy against breast cancer.
Asunto(s)
Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Fragmentos de Péptidos/farmacología , Proteínas de Unión al ARN/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción , Transcripción Genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cellular unfolded protein response (UPR) is induced when endoplasmic reticulum (ER) is under stress. XBP-1S, the active isoform of X-box binding protein 1 (XBP-1), is a key regulator of UPR. Previously, we showed that a histone acetyltransferase (HAT), p300/CBP-associated factor (PCAF), binds to XBP-1S and functions as an activator of XBP-1S. Here, we identify general control nonderepressible 5 (GCN5), a HAT with 73% identity to PCAF, as a novel XBP-1S regulator. Both PCAF and GCN5 bind to the same domain of XBP-1S. Surprisingly, GCN5 potently blocks the XBP-1S-mediated transcription, including cellular UPR genes and latent membrane protein 1 of Epstein-Barr virus. Unlike PCAF, GCN5 acetylates XBP-1S and enhances nuclear retention and protein stability of XBP-1S. However, such GCN5-mediated acetylation of XBP-1S shows no effects on XBP-1S activity. In addition, the HAT activity of GCN5 is not required for repression of XBP-1S target genes. We further demonstrate that GCN5 inhibits XBP-1S-mediated transcription by disrupting the PCAF-XBP-1S interaction and preventing the recruitment of XBP-1S to its target genes. Taken together, our results represent the first work demonstrating that GCN5 and PCAF exhibit different functions and antagonistically regulate the XBP-1S-mediated transcription.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Factores de Transcripción/biosíntesis , Activación Transcripcional/fisiología , Factores de Transcripción p300-CBP/metabolismo , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Humanos , Inmunoprecipitación , Reacción en Cadena de la Polimerasa , Factores de Transcripción del Factor Regulador X , Transcripción Genética , Transfección , Proteína 1 de Unión a la X-BoxRESUMEN
Bromodomain-containing protein 4 (Brd4) and hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) are two opposing regulators of the positive transcription elongation factor b (P-TEFb), which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb's transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis. Here we will discuss the current knowledge on Brd4 and HEXIM1 and their implication as novel therapeutic options against cancer.
Asunto(s)
Neoplasias/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Factores de Transcripción/químicaRESUMEN
Expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes is regulated by a strong enhancer-containing promoter with multiple binding sites for various transcription factors, including cyclic AMP response element binding protein 1 (CREB1). Here we show that overexpression of CREB1 potently blocked MIE transcription and HCMV replication. Surprisingly, CREB1 still exhibited strong inhibition of the MIE promoter when all five CREB binding sites within the enhancer were mutated, suggesting that CREB1 regulated the MIE gene expression indirectly. Promoter deletion analysis and site-directed mutagenesis identified the region between -130 and -50 upstream of the transcription start site of the MIE gene as the "CREB1 responsive region". Mutations of SP1/3 and NF-κB binding sites within this region interrupted the inhibitory effect induced by CREB1 overexpression. Our findings suggest that overexpression of CREB1 can cause repression of HCMV replication and may contribute to the development of new anti-HCMV strategies.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Citomegalovirus/fisiología , Expresión Génica , Interacciones Huésped-Patógeno , Replicación Viral , Línea Celular , Citomegalovirus/genética , Análisis Mutacional de ADN , ADN Viral/genética , Genes Inmediatos-Precoces , Humanos , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation of RNA polymerase II and Tat transactivation of human immunodeficiency virus. Besides P-TEFb, several proteins have been identified as HEXIM1 binding proteins. It is noteworthy that more than half of the HEXIM1 binding partners are involved in cancers. P53 and two key regulators of the p53 pathway, nucleophosmin (NPM) and human double minute-2 protein (HDM2), are among the factors identified. This review will focus on the functional importance of the interactions between HEXIM1 and p53/NPM/HDM2. NPM and the cytoplasmic mutant of NPM, NPMc+, were found to regulate P-TEFb activity and RNA polymerase II transcription through the interaction with HEXIM1. Importantly, more than one-third of acute myeloid leukemia (AML) patients carry NPMc+, suggesting the involvement of HEXIM1 in tumorigenesis of AML. HDM2 was found to ubiquitinate HEXIM1. The HDM2-mediated ubiquitination of HEXIM1 did not lead to protein degradation of HEXIM1 but enhanced its inhibitory activity on P-TEFb. Recently, HEXIM1 was identified as a novel positive regulator of p53. HEXIM1 prevented p53 ubiquitination by competing with HDM2 in binding to p53. Taken together, the new evidence suggests a role of HEXIM1 in regulating the p53 pathway and tumorigenesis.
RESUMEN
Hexamethylene bisacetamide inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9)/cyclin T1. P-TEFb is an essential regulator for the transcriptional elongation by RNA polymerase II. A genome-wide study using human embryonic stem cells shows that most mRNA synthesis is regulated at the stage of transcription elongation, suggesting a possible role for P-TEFb/HEXIM1 in the gene regulation of stem cells. In this report, we detected a marked increase in HEXIM1 protein levels in the differentiated human pluripotent stem cells (hPSCs) induced by LY294002 treatment. Since no changes in CDK9 and cyclin T1 were observed in the LY294002-treated cells, increased levels of HEXIM1 might lead to inhibition of P-TEFb activity. However, treatment with a potent P-TEFb inhibiting compound, flavopiridol, failed to induce hPSC differentiation, ruling out the possible requirement for P-TEFb kinase activity in hPSC differentiation. Conversely, differentiation was observed when hPSCs were incubated with hexamethylene bisacetamide, a HEXIM1 inducing reagent. The involvement of HEXIM1 in the regulation of hPSCs was further supported when overexpression of HEXIM1 concomitantly induced hPSC differentiation. Collectively, our study demonstrates a novel role of HEXIM1 in regulating hPSC fate through a P-TEFb-independent pathway.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas de Unión al ARN/metabolismo , Acetamidas/farmacología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , Ectodermo/citología , Flavonoides/farmacología , Humanos , Mesodermo/citología , Piperidinas/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Factor B de Elongación Transcripcional Positiva/metabolismo , Factores de Transcripción , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Pin1 isomerizes the phosphorylated Ser/Thr-Pro peptide bonds and regulates the functions of its binding proteins by inducing conformational changes. Involvement of Pin1 in the aging process has been suggested based on the phenotype of Pin1-knockout mice and its interaction with lifespan regulator protein, p66 (Shc) . In this study, we utilize a proteomic approach and identify peroxiredoxin 1 (PRDX1), another regulator of aging, as a novel Pin1 binding protein. Pin1 binds to PRDX1 through interacting with the phospho-Thr ( 90) -Pro ( 91) motif of PRDX1, and this interaction is abolished when the Thr ( 90) of PRDX1 is mutated. The Pin1 binding motif, Thr-Pro, is conserved in the 2-Cys PRDXs, PRDX1-4 and the interactions between Pin1 and PRDX2-4 are also demonstrated. An increase in hydrogen peroxide buildup and a decrease in the peroxidase activity of 2-Cys PRDXs were observed in Pin1 (-/-) mouse embryonic fibroblasts (MEFs), with the activity of PRDXs restored when Pin1 was re-introduced into the cells. Phosphorylation of PRDX1 at Thr ( 90) has been shown to inhibit its peroxidase activity; however, how exactly the activity of PRDX1 is regulated by phosphorylation still remains unknown. Here, we demonstrate that Pin1 facilitates the protein phosphatase 2A-mediated dephosphorylation of PRDX1, which helps to explain the accumulation of the inactive phosphorylated form of PRDX1 in Pin1 (-/-) MEFs. Collectively, we identify Pin1 as a novel PRDX1 binding protein and propose a mechanism for Pin1 in regulating the metabolism of reactive oxygen species in cells.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Peroxirredoxinas/metabolismo , Envejecimiento , Animales , Células HEK293 , Células HeLa , Humanos , Ratones , Peptidilprolil Isomerasa de Interacción con NIMA , Oxidación-Reducción , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Nucleophosmin (NPM), an important regulator in p53 signaling pathway, is one of the most frequently mutated genes in acute myeloid leukemia (AML). In our previous study, we found that hexamethylene bisacetamide inducible protein 1 (HEXIM1) interacted with both wild-type NPM and cytoplasmic-misallocated NPMc(+) mutant, leading to an increase in RNA polymerase II transcription. Here, we examine the protein expression in wild-type NPM (AML2) and NPMc(+) mutant (AML3) AML cell lines. Significant lower levels of NPM, HEXIM1 and p53 proteins are detected in AML3 cells, and such differential protein expression is not regulated at transcriptional or post-translational stages. Effects of several anticancer compounds on cell viability of AML2 and AML3 cells are investigated. Compared to AML3 cells, AML2 cells are more sensitive to the treatment of the DNA-damaging compounds (doxorubicin and etoposide) and a specific p53-inducing compound (nutlin-3). However, no significant difference in cytotoxicity was observed when AML2 and AML3 cells were treated with cyclin-dependent kinase inhibitors, flavopiridol and CYC202. Our results provide a novel insight into the functional impact of the NPMc(+) mutation on protein expression and the potential approaches for selective therapy of AML.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/patología , Proteínas Nucleares/análisis , Proteína p53 Supresora de Tumor/análisis , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Expresión Génica , Humanos , Mutación , Nucleofosmina , Proteínas de Unión al ARN/análisis , Factores de TranscripciónRESUMEN
The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter.
Asunto(s)
Citomegalovirus/genética , Deltaretrovirus/química , Productos del Gen tax/fisiología , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/biosíntesis , Deltaretrovirus/genética , Productos del Gen tax/genética , Genes Inmediatos-Precoces , Células HEK293 , Humanos , Transactivadores/genética , Transgenes , Proteínas Virales/genéticaRESUMEN
Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells.
Asunto(s)
Luciferasas/genética , Ingeniería de Proteínas/métodos , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células CHO , Procesos de Crecimiento Celular/fisiología , Línea Celular , Cricetinae , Cricetulus , Eritropoyetina/genética , Eritropoyetina/metabolismo , Humanos , Interferón gamma/genética , Interferón gamma/metabolismo , Luciferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is an inhibitor of the positive transcription elongation factor b (P-TEFb), which controls RNA polymerase II transcription and human immunodeficiency virus Tat transactivation. In cells, more than half of P-TEFb is associated with HEXIM1 resulting in the inactivation of P-TEFb. Recently, we found that nucleophosmin (NPM), a key factor involved in p53 signaling pathway, interacts with HEXIM1 and activates P-TEFb-dependent transcription. Here we report that human double minute-2 protein (HDM2), a p53-specific E3 ubiquitin ligase, specifically ubiquitinates HEXIM1 through the lysine residues located within the basic region of HEXIM1. However, the HDM2-induced HEXIM1 ubiquitination does not lead to proteasome-mediated protein degradation. Fusion of ubiquitin to HEXIM1 demonstrates stronger inhibition on P-TEFb-dependent transcription. Our results demonstrate that HDM2 functions as a specific E3 ubiquitin ligase for HEXIM1, suggesting a possible role for HEXIM1 ubiquitination in the regulation of P-TEFb activity.