RESUMEN
We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Animales , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Péptido Hidrolasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be â¼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with â¼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a â¼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.
Asunto(s)
Antineoplásicos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Imidazoles , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Piridinas , PirimidinasRESUMEN
PURPOSE: The EphA2 tyrosine kinase is frequently overexpressed in human tumors that are also treated with radiation. However, few studies have examined the effect of radiation on the EphA2 receptor itself. The purpose of this project was to investigate the impact of radiation on EphA2 to better understand mechanisms of radioresistance. MATERIALS AND METHODS: Cell lines were exposed to X-rays and assayed for changes in EphA2 protein levels and phosphorylation over time by Western blotting. HEK293 cells stably expressing wild-type EphA2 or the S897A mutant were analyzed for cell survival from X-rays. RESULTS: Treatment of different cancer cell lines with 2 Gy of X-rays induced the phosphorylation of EphA2 on S897 but no changes were found in EphA2 total levels or its tyrosine phosphorylation. Radiation-induced S897 phosphorylation was unaffected by an AKT inhibitor but blocked by a MEK or RSK inhibitor. HEK293 cells expressing the EphA2 S897A mutant had a nearly 2-fold lower level of cell survival from X-rays than cells expressing wild-type EphA2. CONCLUSIONS: These findings show that radiation induces S897 EphA2 phosphorylation, an event associated with increased cell survival. Therefore, targeting pathways that mediate EphA2 S897 phosphorylation may be a beneficial strategy to reduce radioresistance.
Asunto(s)
Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Sistema de Señalización de MAP Quinasas/fisiología , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Fosfoserina/metabolismo , Radiación Ionizante , Receptor EphA2/metabolismo , Relación Dosis-Respuesta en la Radiación , Células HEK293 , Humanos , Fosforilación/efectos de la radiación , Dosis de Radiación , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
As core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.
Asunto(s)
Proteínas Argonautas/metabolismo , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , MicroARNs/genética , Proteínas Argonautas/genética , Autoantígenos/metabolismo , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/química , Proteínas con Dominio LIM/genética , MicroARNs/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
In addition to playing roles in the genesis and progression of cancer, mutant p53 also appears to play a significant role in the response to cancer therapy. In response to chemotherapy and radiation, two mainstays of cancer treatment, most cancer cells harboring p53 mutations show a reduced sensitivity compared to cells lacking p53 or those with wild type p53. However, there are also many instances where mutant p53 has shown no effect or enhances cellular sensitivity to chemotherapy and radiation. Similar to the in vitro cellular studies, the majority of clinical studies show a correlation between the presence of mutant p53 in patient tumors and adverse outcomes following treatment with chemotherapy agents or radiation in comparison to tumors with wild-type p53. However, it still remains unclear whether the presence of mutant p53 in tumors can serve as a reliable prognostic factor and aid in treatment planning. Thus, as genomic analysis of patient tumors becomes more cost effective, the role of mutant p53 in tumor responses from cancer therapy ultimately needs to be addressed. This chapter will discuss current mechanisms of how p53 mutations affect cellular responses to chemotherapy and radiation and discuss patient outcomes based on p53 status.
Asunto(s)
Genes p53 , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/radioterapia , Antineoplásicos/uso terapéutico , Rayos gamma , Humanos , Neoplasias/genéticaRESUMEN
Protein-protein interaction studies can provide valuable insight into protein function. One of the most practical and high-yielding approaches is immunoprecipitation of a bait protein followed by mass spectrometry to identify co-precipitating proteins. Here we describe an effective and simplified version of this method that can be performed in most laboratories using standard laboratory equipment (apart from the mass spectrometer). We further demonstrate the utility of this method to identify proteins that specifically interact with mutant forms of the tumor suppressor protein, p53.
Asunto(s)
Proteínas Mutantes/aislamiento & purificación , Proteómica/métodos , Proteína p53 Supresora de Tumor/aislamiento & purificación , Western Blotting , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoprecipitación , Espectrometría de Masas , Proteínas Mutantes/genética , Mutación , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Cancer cells with p53 mutations, in general, grow more aggressively than those with wild-type p53 and show "gain of function" (GOF) phenotypes such as increased growth rate, enhanced resistance to chemotherapeutic drugs, increased cell motility and tumorigenicity; although the mechanism for this function remains unknown. In this communication we report that p53-mediated NF-κB2 up-regulation significantly contributes to the aggressive oncogenic behavior of cancer cells. Lowering the level of mutant p53 in a number of cancer cell lines resulted in a loss of GOF phenotypes directly implicating p53 mutants in the process. RNAi against NF-κB2 in naturally occurring cancer cell lines also lowers GOF activities. In H1299 cells expressing mutant p53, chromatin immunoprecipitation (ChIP) assays indicate that mutant p53 induces histone acetylation at specific sites on the regulatory regions of its target genes. ChIP assays using antibodies against transcription factors putatively capable of interacting with the NF-κB2 promoter show increased interaction of CBP and STAT2 in the presence of mutant p53. Thus, we propose that in H1299 cells, mutant p53 elevates expression of genes capable of enhancing cell proliferation, motility, and tumorigenicity by inducing acetylation of histones via recruitment of CBP and STAT2 on the promoters causing CBP-mediated histone acetylation.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Mutación , Subunidad p52 de NF-kappa B/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción STAT/metabolismo , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Ratones , Subunidad p52 de NF-kappa B/deficiencia , Subunidad p52 de NF-kappa B/metabolismo , Unión Proteica/genética , Interferencia de ARN , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genéticaRESUMEN
The p53 gene is one of the most frequently mutated genes in human cancer. Some p53 mutations impart additional functions that promote oncogenesis. To investigate how these p53 mutants function, a proteomic analysis was performed. The protein, translocator of the inner mitochondrial membrane 50 (Tim50), was upregulated in a non-small cell lung carcinoma cell line (H1299) that expressed the p53 mutants R175H and R273H compared to cells lacking p53. Tim50 was also elevated in the breast cancer cell lines MDA-MB-468 and SK-BR-3, that endogenously express the p53 mutants R175H and R273H, respectively, compared to MCF-10A. The p53 mutants R175H and R273H, but not WT p53, upregulated the expression of a Tim50 promoter construct and chromatin immunoprecipitation (ChIP) analysis indicated increased histone acetylation and increased interaction of the transcription factors Ets-1, CREB and CREB-binding protein (CBP) with the Tim50 promoter in the presence of mutant p53. Finally, reduction of Tim50 expression reduced the growth rate and chemoresistance of cells harboring mutant p53 but had no effect upon cells lacking p53. Taken together, these findings identify the Tim50 gene as a transcriptional target of mutant p53 and suggest a novel mechanism by which p53 mutants enhance cell growth and chemoresistance.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Mutación , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/genética , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Regulación hacia ArribaRESUMEN
Models for exploring tyrosine nitration in proteins have been created based on 3D structural features of 20 proteins for which high-resolution X-ray crystallographic or NMR data are available and for which nitration of 35 total tyrosines has been experimentally proven under oxidative stress. Factors suggested in previous work to enhance nitration were examined with quantitative structural descriptors. The role of neighboring acidic and basic residues is complex: for the majority of tyrosines that are nitrated the distance to the heteroatom of the closest charged side chain corresponds to the distance needed for suspected nitrating species to form hydrogen bond bridges between the tyrosine and that charged amino acid. This suggests that such bridges play a very important role in tyrosine nitration. Nitration is generally hindered for tyrosines that are buried and for those tyrosines for which there is insufficient space for the nitro group. For in vitro nitration, closed environments with nearby heteroatoms or unsaturated centers that can stabilize radicals are somewhat favored. Four quantitative structure-based models, depending on the conditions of nitration, have been developed for predicting site-specific tyrosine nitration. The best model, relevant for both in vitro and in vivo cases, predicts 30 of 35 tyrosine nitrations (positive predictive value) and has a sensitivity of 60/71 (11 false positives).
Asunto(s)
Modelos Moleculares , Nitratos/química , Nitritos/química , Nitrógeno/química , Tirosina/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Enlace de Hidrógeno , Imagen por Resonancia Magnética , Modelos Químicos , Estructura Molecular , Estrés Oxidativo , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The goal of radiation therapy is to reduce or eliminate tumor burden while sparing normal tissues from long-term injury. Thoracic radiation presents a unique challenge because of the inherent sensitivity of normal lung tissue to radiation. Damage to normal lung tissue presents a major obstacle in the treatment of individuals. To overcome this problem, a number of strategies are being used, including the modulation of dose volume, the use of image-guided radiotherapy, and the use of agents designed to reduce lung injury from radiation. Herein we discuss our current knowledge of the molecular and cellular events that occur after radiation therapy, the clinical manifestations of radiation-induced lung injury, current strategies to minimize lung injury, and recent experimental methods to reduce lung injury and their potential for translation into the clinic.
Asunto(s)
Neoplasias Pulmonares/radioterapia , Neumonitis por Radiación/etiología , Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia Conformacional/efectos adversos , Relación Dosis-Respuesta en la Radiación , Humanos , Pulmón/efectos de la radiación , Dosificación Radioterapéutica , Radioterapia Asistida por Computador/métodos , Radioterapia Conformacional/métodosRESUMEN
PURPOSE: To identify temporal changes in protein expression in the irradiated rat lung and generate putative mechanisms underlying the radioprotective effect of the manganese superoxide dismutase mimetic MnTE-2-PyP(5+). METHODS AND MATERIALS: Female Fischer 344 rats were irradiated to the right hemithorax with a single dose of 28 Gy and killed from day 1 to 20 weeks after irradiation. Proteomic profiling was performed to identify proteins that underwent significant changes in abundance. Some irradiated rats were administered MnTE-2-PyP(5+) and changes in protein expression and phosphorylation determined at 6 weeks after irradiation. RESULTS: Radiation induced a biphasic stress response in the lung, as shown by the induction of heme oxygenase 1 at 1-3 days and at 6-8 weeks after irradiation. At 6-8 weeks after irradiation, the down-regulation of proteins involved in cytoskeletal architecture (filamin A and talin), antioxidant defense (biliverdin reductase and peroxiredoxin II), and cell signaling (ß-catenin, annexin II, and Rho-guanosine diphosphate dissociation inhibitor) was observed. Treatment with MnTE-2-PyP(5+) partially prevented the apparent degradation of filamin and talin, reduced the level of cleaved caspases 3 and 9, and promoted Akt phosphorylation as well as ß-catenin expression. CONCLUSION: A significant down-regulation of proteins and an increase in protein markers of apoptosis were observed at the onset of lung injury in the irradiated rat lung. Treatment with MnTE-2-PyP(5+), which has been demonstrated to reduce lung injury from radiation, reduced apparent protein degradation and apoptosis indicators, suggesting that preservation of lung structural integrity and prevention of cell loss may underlie the radioprotective effect of this compound.
Asunto(s)
Pulmón/efectos de la radiación , Metaloporfirinas/farmacología , Proteínas/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Protectores contra Radiación/farmacología , Animales , Anexina A2/metabolismo , Apoptosis , Caspasa 9/metabolismo , Proteínas Contráctiles/metabolismo , Regulación hacia Abajo , Femenino , Filaminas , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Hemo-Oxigenasa 1/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Proteínas de Microfilamentos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Peroxirredoxinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Dosis de Radiación , Traumatismos Experimentales por Radiación/prevención & control , Ratas , Ratas Endogámicas F344 , Talina/metabolismo , Factores de Tiempo , beta Catenina/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Previous studies demonstrate that nitric oxide (NO) promotes p53 transcriptional activity by a classical DNA damage responsive mechanism involving activation of ATM/ATR and phosphorylation of p53. These studies intentionally used high doses of NO donors to achieve the maximum DNA damage. However, lower concentrations of NO donors also stimulate rapid and unequivocal nuclear retention of p53 but apparently do not require ATM/ATR-dependent p53 phosphorylation or total p53 protein accumulation. To identify possible mechanisms for p53 activation at low NO levels, the role of Tyr nitration in p53 activation was evaluated. Low concentrations of the NO donor, DETA NONOate (<200 microM), exclusively nitrate Tyr327 within the tetramerization domain promoting p53 oligomerization, nuclear accumulation, and increased DNA-binding activity without p53 Ser15 phosphorylation. Molecular modeling indicates that nitration of one Tyr327 stabilizes the dimer by about 2.67 kcal mol(-1). Significant quantitative and qualitative differences in the patterns of p53-target gene modulation by low (50 microM), non-DNA-damaging and high (500 microM), DNA-damaging NO donor concentrations were shown. These results demonstrate a new posttranslational mechanism for modulating p53 transcriptional activity responsive to low NO concentrations and independent of DNA damage signaling.
Asunto(s)
Biopolímeros/metabolismo , Nitratos/metabolismo , Donantes de Óxido Nítrico/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Humanos , Espectrometría de Masas , Modelos Moleculares , Óxido Nítrico Sintasa/metabolismo , Fosforilación , Termodinámica , Proteína p53 Supresora de Tumor/químicaRESUMEN
microRNAs are a class of small RNA molecules that associate with Argonaute proteins to regulate gene expression. Argonaute proteins not only mediate the functions of microRNAs, but also play an essential role in the biogenesis of microRNAs. Here, we report that stable, long-term overexpression of Argonaute2, an Argonaute isoform, induces the production of a number of microRNAs, such as the let-7 family of microRNAs, in 293T cells. On the other hand, the expression of many microRNAs is insensitive to elevated Argonaute levels, and microRNAs in the miR-17-92 and homologous clusters are even down-regulated. The down-regulation may result from the let-7-mediated inhibition of the expression of Myc, which positively controls the transcription of the microRNAs clusters. Our data suggest that human cells have a mechanism for microRNA homeostasis, and that overexpression of a general microRNA processing factor can differentially regulate the expression of specific microRNAs.
Asunto(s)
Factor 2 Eucariótico de Iniciación/fisiología , Regulación de la Expresión Génica , MicroARNs/biosíntesis , Proteínas Argonautas , Northern Blotting , Línea Celular , Factor 2 Eucariótico de Iniciación/genética , Humanos , Immunoblotting , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Interferencia de ARN/fisiologíaRESUMEN
Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Antracenos/farmacología , Proteínas Argonautas , Arsenitos/farmacología , Western Blotting , Línea Celular , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Flavonoides/farmacología , Glutatión Transferasa , Humanos , Imidazoles/farmacología , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Espectrometría de Masas , Microscopía Confocal , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Mutación , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Serina/genética , Compuestos de Sodio/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The NF-kappaB family of transcription factors is an important component of stress-activated cytoprotective signal transduction pathways. Previous studies demonstrated that some activation mechanisms require phosphorylation, ubiquitination, and degradation of the inhibitor protein, IkappaBalpha. Herein, it is demonstrated that ionizing radiation in the therapeutic dose range stimulates NF-kappaB activity by a mechanism in which IkappaBalpha tyrosine 181 is nitrated as a consequence of constitutive NO* synthase activation, leading to dissociation of intact IkappaBalpha from NF-kappaB. This mechanism does not appear to require IkappaBalpha kinase-dependent phosphorylation or proteolytic degradation of IkappaBalpha. Tyrosine 181 is involved in several noncovalent interactions with the p50 subunit of NF-kappaB stabilizing the IkappaBalpha-NF-kappaB complex. Evaluation of hydropathic interactions of the IkappaBalpha-p50 complex on the basis of the crystal structure of the complex is consistent with nitration disrupting these interactions and dissociating the IkappaBalpha-NF-kappaB complex. Tyrosine nitration is not commonly studied in the context of signal transduction. However, these results indicate that tyrosine nitration is an important post-translational regulatory modification for NF-kappaB activation and possibly for other signaling molecules modulated by mild and transient oxidative and nitrosative stresses.
Asunto(s)
Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , Nitrógeno/metabolismo , Tirosina/metabolismo , Animales , Neoplasias de la Mama/patología , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Relación Dosis-Respuesta en la Radiación , Femenino , Genes Reporteros , Humanos , Cinética , Luciferasas/metabolismo , Modelos Moleculares , Mutación , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/genética , Resonancia Magnética Nuclear Biomolecular , Oxidantes/farmacología , Ácido Peroxinitroso/farmacología , Procesamiento Proteico-Postraduccional , Teoría Cuántica , ARN Interferente Pequeño/metabolismo , Radiación Ionizante , Transfección , Tirosina/análogos & derivados , Tirosina/química , Tirosina/genéticaRESUMEN
Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.
Asunto(s)
Ligasas de Carbono-Nitrógeno/biosíntesis , Regulación Enzimológica de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Glucógeno Sintasa Quinasa 3 beta , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosforilación , Homología de Secuencia de Aminoácido , Serina/químicaRESUMEN
Clostridium taeniosporum spores have about 12 large, flat, ribbon-like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the 'encasement'. Appendages are about 4.5 microm long, 0.5 microm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair-like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen-like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative sigma(K)-dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.
Asunto(s)
Clostridium/citología , Clostridium/fisiología , Genes Bacterianos , Esporas Bacterianas/ultraestructura , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cromosomas Bacterianos , Clostridium/química , Clostridium/genética , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Esporas Bacterianas/química , Esporas Bacterianas/genéticaRESUMEN
Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase implicated in cell death and smooth muscle contractility, but its mechanism of regulation is unknown. We have identified six phosphorylation sites in ZIPK that regulate both its enzyme activity and localization, including Thr180, Thr225, Thr265, Thr299, Thr306, and Ser311. Mutational analysis showed that phosphorylation of Thr180 in the kinase activation T-loop, Thr225 in the substrate-binding groove, and Thr265 in kinase subdomain X is essential for full ZIPK autophosphorylation and activity toward exogenous substrates. Abrogation of phosphorylation of Thr299, Thr306, and Ser311 had little effect on enzyme activity, but mutation of Thr299 and Thr300 to alanine resulted in redistribution of ZIPK from the cytosol to the nucleus. Mutation of Thr299 alone to alanine caused ZIPK to assume a diffuse cellular localization, whereas T299D redistributed the enzyme to the cytoplasm. C-terminal truncations of ZIPK at amino acid 273 or 342 or mutation of the leucine zipper motif increased ZIPK activity toward exogenous substrates by severalfold, suggesting a phosphorylation-independent autoinhibitory role for the C-terminal domain. Additionally, mutation of the leucine zipper reduced the ability of ZIPK to oligomerize and also caused ZIPK to relocalize from the cytoplasm to the nucleus in vivo. Together, our findings show that ZIPK is positively regulated by phosphorylation within its kinase domain and that it contains an inhibitory C-terminal domain that controls enzyme activity, localization, and oligomerization.