RESUMO
YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Fosfoproteínas/química , Fosfoproteínas/efeitos da radiação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efeitos da radiação , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Bacillus subtilis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Difusão Dinâmica da Luz , Luz , Modelos Moleculares , Fosfoproteínas/metabolismo , Processos Fotoquímicos , Fotorreceptores Microbianos/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos da radiação , Estrutura Quaternária de Proteína/efeitos da radiaçãoRESUMO
During space travel astronauts will be exposed to a very low, mixed field of radiation containing different high LET particles of varying energies, over an extended period. Thus, defining how human cells respond to these complex low dose exposures is important in ascertaining risk. In the current study, we have chosen to investigate how low doses of three different ion's at various energies uniquely change the kinetics of three different phospho-proteins. A normal hTERT immortalized fibroblast cell line, 82-6, was exposed to a range of lower doses (0.05-0.5 Gy) of radiation of different qualities and energies (Si 1000 MeV/u, Si 300 MeV/u, Si 173 MeV/u, Si 93 MeV/u, Fe 1000 MeV/u, Fe 600 MeV/u, Fe 300 MeV/u, Ti 300 MeV/u, Ti 326 MeV/u, Ti 386 MeV/u), covering a wide span of LET's. Exposed samples were analyzed for the average intensity of signal as a fold over the geometric mean level of the sham controls. Three phospho-proteins known to localize to DNA DSBs following radiation (γH2AX, pATF2, pSMC1) were studied. The kinetics of their response was quantified by flow cytometery at 2 and 24 h post exposure. These studies reveal unique kinetic patterns based on the ion, energy, fluence and time following exposure. In addition, γH2AX phosphorylation patterns are uniquely different from phospho-proteins known to be primarily phosphorylated by ATM. This latter finding suggests that the activating kinase(s), or the phosphatases deactivating these proteins, exhibit differences in their response to various radiation qualities and/ or doses of exposure. Further studies will be needed to better define what the differing kinetics for the kinases activated by the unique radiation qualities plays in the biological effectiveness of the particle.
Assuntos
Íons Pesados , Transferência Linear de Energia , Fosfoproteínas/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Linhagem Celular , Dano ao DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Humanos , Fosforilação/efeitos da radiaçãoRESUMO
Ultraviolet (UV)-B radiation acts as an elicitor to enhance the production of secondary metabolites in medicinal plants. To investigate the mechanisms, which lead to secondary metabolites in Catharanthus roseus under UVB radiation, a phosphoproteomic technique was used. ATP content increased in the leaves of C. roseus under UVB radiation. Phosphoproteins related to calcium such as calmodulin, calcium-dependent kinase, and heat shock proteins increased. Phosphoproteins related to protein synthesis/modification/degradation and signaling intensively changed. Metabolomic analysis indicated that the metabolites classified with pentoses, aromatic amino acids, and phenylpropanoids accumulated under UVB radiation. Phosphoproteomic and immunoblot analyses indicated that proteins related to glycolysis and the reactive-oxygen species scavenging system were changed under UVB radiation. These results suggest that UVB radiation activates the calcium-related pathway and reactive-oxygen species scavenging system in C. roseus. These changes lead to the upregulation of proteins, which are responsible for the redox reactions in secondary metabolism and are important for the accumulation of secondary metabolites in C. roseus under UVB radiation.
Assuntos
Catharanthus/metabolismo , Fosfoproteínas/genética , Proteínas de Plantas/metabolismo , Metabolismo Secundário/efeitos da radiação , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Catharanthus/genética , Catharanthus/efeitos da radiação , Fosfoproteínas/efeitos da radiação , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/efeitos da radiação , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , Plantas Medicinais/efeitos da radiação , Metabolismo Secundário/genética , Transdução de Sinais/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.
Assuntos
Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Neoplasias Encefálicas/terapia , Dano ao DNA/efeitos dos fármacos , Melanoma Experimental/terapia , Radiocirurgia/métodos , Fator de Transcrição STAT3/efeitos dos fármacos , Actinas/efeitos dos fármacos , Actinas/metabolismo , Actinas/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Western Blotting , Neoplasias Encefálicas/secundário , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 3/efeitos da radiação , Linhagem Celular Tumoral , Terapia Combinada , Dano ao DNA/efeitos da radiação , Técnicas In Vitro , Janus Quinase 2/efeitos dos fármacos , Janus Quinase 2/metabolismo , Janus Quinase 2/efeitos da radiação , Melanoma Experimental/secundário , Camundongos , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/efeitos da radiação , Survivina/efeitos dos fármacos , Survivina/metabolismo , Survivina/efeitos da radiação , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/efeitos da radiaçãoRESUMO
Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12-18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-ß by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-ß production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/efeitos da radiação , Neoplasias Mamárias Animais/genética , Neoplasias/imunologia , Neoplasias/radioterapia , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Animais , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linfócitos T CD8-Positivos/efeitos da radiação , Antígeno CTLA-4/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos da radiação , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunoterapia/métodos , Interferon beta/metabolismo , Interferon beta/efeitos da radiação , Neoplasias Mamárias Animais/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nucleotidiltransferases/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Proteínas Repressoras/imunologiaRESUMO
OBJECTIVES: GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. MATERIALS AND METHODS: The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14 days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21 days. RESULTS: The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. CONCLUSION: GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.
Assuntos
Dentina Secundária/metabolismo , Dentina Secundária/efeitos da radiação , Proteínas da Matriz Extracelular/biossíntese , Lasers Semicondutores , Dente Molar/efeitos da radiação , Osteopontina/biossíntese , Fosfoproteínas/biossíntese , Animais , Polpa Dentária/citologia , Polpa Dentária/fisiologia , Proteínas da Matriz Extracelular/efeitos da radiação , Proteínas de Choque Térmico HSP27/biossíntese , Imuno-Histoquímica , Masculino , Dente Molar/citologia , Dente Molar/metabolismo , Odontoblastos/metabolismo , Odontoblastos/efeitos da radiação , Osteopontina/efeitos da radiação , Fosfoproteínas/efeitos da radiação , Ratos , Ratos Wistar , Regulação para Cima/efeitos da radiaçãoRESUMO
Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to â¼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox.
Assuntos
Fosfoproteínas/análise , Fosfoproteínas/efeitos da radiação , Fotólise/efeitos da radiação , Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Raios Ultravioleta , Cromatografia Líquida/instrumentaçãoRESUMO
Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aß strand of LOV2 (A'α/Aß gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aß gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/efeitos da radiação , Luz , Fosfoproteínas/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Cisteína/química , Ativação Enzimática/efeitos da radiação , Mononucleotídeo de Flavina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/efeitos da radiação , Fosforilação/efeitos da radiação , Fotoquímica , Conformação Proteica , Proteínas Serina-Treonina Quinases , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: The aim of this study was to evaluate the effects of infrared LED (850nm) irradiation on dentin matrix proteins expression and synthesis by cultured stem cells from human exfoliated deciduous teeth (SHED). METHODS: Near-exfoliation primary teeth were extracted (n=3), and SHED cultures were characterized by immunofluorescence using STRO-1, CD44, CD146, Nanog and OCT3/4 antibodies, before experimental protocol. The SHEDs were seeded (3×10(4) cells/cm(2)) with DMEM containing 10% FBS. After 24-h incubation, the culture medium was replaced by osteogenic differentiation medium, and the cells were irradiated with LED light at energy densities (EDs) of 0 (control), 2, or 4J/cm(2) (n=8). The irradiated SHEDs were then evaluated for alkaline phosphatase (ALP) activity, total protein (TP) production, and collagen synthesis (SIRCOL™ Assay), as well as ALP, collagen type I (Col I), dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein (DMP-1) gene expression (qPCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). RESULTS: Increased ALP activity and collagen synthesis, as well as gene expression of DSPP and ALP, were observed for both EDs compared with non-irradiated cells. The ED of 4J/cm(2) also increased gene expression of COL I and DMP-1. CONCLUSIONS: In conclusion, infrared LED irradiation was capable of biostimulating SHEDs by increasing the expression and synthesis of proteins related with mineralized tissue formation, with overall better results for the energy dose of 4J/cm(2). CLINICAL SIGNIFICANCE: Phototherapy is an additional approach for the clinical application of LED in Restorative Dentistry. Infrared LED irradiation of the cavity's floor could biostimulate subjacent pulp cells, improving local tissue healing.
Assuntos
Polpa Dentária/citologia , Proteínas da Matriz Extracelular/efeitos da radiação , Fototerapia/métodos , Células-Tronco/efeitos da radiação , Dente Decíduo/citologia , Fosfatase Alcalina/análise , Fosfatase Alcalina/efeitos da radiação , Técnicas de Cultura de Células , Células Cultivadas , Colágeno Tipo I/análise , Colágeno Tipo I/efeitos da radiação , Meios de Cultura , Proteínas da Matriz Extracelular/análise , Humanos , Raios Infravermelhos , Osteogênese/efeitos da radiação , Fosfoproteínas/análise , Fosfoproteínas/efeitos da radiação , Proteínas/análise , Proteínas/efeitos da radiação , Doses de Radiação , Sialoglicoproteínas/análise , Sialoglicoproteínas/efeitos da radiação , Esfoliação de Dente , Regulação para CimaRESUMO
Cyanobacterial phytochrome 1 (Cph1) is a red/far-red light regulated histidine kinase, which together with its response regulator (Rcp1) forms a two-component light signaling system in Synechocystis 6803. In the present study we followed the in vitro autophosphorylation of Cph1 and the subsequent phosphotransfer to Rcp1 in different ionic milieus and following different light treatments. Both processes were red/far-red reversible with activity manifested in the Pr ground state (in darkness or after far-red irradiation) and with strongest activities being exhibited in the presence of Mn(2+). In vivo and in vitro assembled holoproteins in the Pr state displayed at least 4-fold higher efficiencies (k(cat)/K(m)) for autophosphorylation and phosphotransfer than the apoprotein or the holoprotein at photoequilibrium in red light. The reduced activities observed following red light treatments were consistent with the Pfr state being enzymatically inactive. Thus, both the rate of kinase autophosphorylation and the rate of phosphotransfer regulate the phosphorylation state of the response regulator, consistent with the rotary switch model regulating accessibility of the histidine target.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Luz , Fitocromo/química , Fitocromo/fisiologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Transdução de Sinais/fisiologia , Synechocystis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Cátions Bivalentes/química , Histidina Quinase , Concentração de Íons de Hidrogênio , Cinética , Manganês/química , Fosfoproteínas/química , Fosfoproteínas/fisiologia , Fosfoproteínas/efeitos da radiação , Fosforilação/efeitos da radiação , Fotorreceptores Microbianos , Fitocromo/efeitos da radiação , Proteínas Quinases/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Synechocystis/efeitos da radiação , Raios UltravioletaRESUMO
Phototropins (phot1 and phot2) are blue-light receptor kinases controlling a range of responses that optimize the photosynthetic efficiency of plants. Light sensing is mediated by two flavin-binding motifs, known as LOV1 and LOV2, located within the N-terminal region of the protein. Photoexcitation via LOV2 leads to activation of the C-terminal kinase domain and consequently receptor autophosphorylation. However, knowledge of the in-vivo phosphorylation sites for Arabidopsis phototropins is lacking and has impeded progress in elucidating the functional significance of receptor phosphorylation. We have purified phot1 from Arabidopsis and identified the in-vivo sites of receptor phosphorylation by liquid chromatography tandem mass spectrometry. Arabidopsis-derived phot1 binds flavin mononucleotide as chromophore and is phosphorylated at four major sites located upstream of LOV2 (Ser(58), Ser(85), Ser(350), and Ser(410)), three of which are induced by blue light. Nevertheless, structure-function analysis indicates that the biological activity of phot1 can be attributed to a modular unit comprising the LOV2-kinase region of the protein. Thus, peptide regions upstream of LOV2, including the sites of receptor phosphorylation identified here, do not appear to be important for receptor signaling. By contrast, these regions may be necessary for maximizing stomatal performance and possibly light-induced relocalization of phot1.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Mapeamento Cromossômico/métodos , Luz , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fotossíntese/efeitos da radiação , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Mononucleotídeo de Flavina/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Espectrometria de Massas , Fosfoproteínas/efeitos da radiação , Fosforilação , Fotossíntese/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos da radiação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiaçãoRESUMO
Pulsed electromagnetic field (PEMF) devices are approved for the healing of bone nonunions, but there is a lack of understanding as to their mechanism of action at the cell and molecular level. Intermittent parathyroid hormone (PTH) therapy is currently utilized for treatment of osteoporosis, and is also being investigated for the purpose of augmenting fracture healing. Insulin and IGF-1 are also thought to play important anabolic roles in osteogenesis. In this report, signaling pathways activated by acute PTH or insulin treatments were compared to those activated by PEMF treatment in osteoblast-like cells. Some signaling molecules like the extracellular response kinases 1/2 (Erk1/2) and the cAMP response element binding protein (CREB) were activated by insulin and PTH, respectively, but not by PEMF treatment. Other signaling molecules like the insulin receptor substrate-1 (IRS-1), the S6 ribosomal subunit kinase, and the endothelial nitric oxide synthase (eNOS) were phosphorylated by PTH, insulin, and PEMF to the same relative extent and within the same time frame. IRS-1, eNOS, and S6 have been implicated in bone anabolism, and our results suggest that the anabolic effects of PEMF may be mediated, in part, through the activation of these proteins.
Assuntos
Anabolizantes/farmacologia , Insulina/farmacologia , Osteoblastos/efeitos da radiação , Hormônio Paratireóideo/farmacologia , Radiação , Transdução de Sinais/efeitos da radiação , Animais , Proteína de Ligação a CREB/efeitos dos fármacos , Proteína de Ligação a CREB/metabolismo , Proteína de Ligação a CREB/efeitos da radiação , Linhagem Celular , Proteínas Substratos do Receptor de Insulina , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos da radiação , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo III/efeitos da radiação , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Fosforilação , Radioterapia , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas/efeitos da radiação , Transdução de Sinais/efeitos dos fármacosRESUMO
A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.
Assuntos
Fibroblastos/efeitos da radiação , Fosfoproteínas/metabolismo , Fosfoproteínas/efeitos da radiação , Proteômica/métodos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos , Sequência de Aminoácidos , Células Cultivadas , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/efeitos da radiação , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Proteína Oncogênica v-akt/metabolismo , Proteína Oncogênica v-akt/efeitos da radiação , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosforilação , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Ribosilação do ADP/genética , Oxirredutases do Álcool , Animais , Nucléolo Celular/metabolismo , Células Cultivadas , Proteínas Correpressoras , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/efeitos da radiação , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos da radiação , Transporte Proteico , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Raios UltravioletaRESUMO
The purpose of this study was to compare the relative amount of low molecular weight salivary proteins in patients with head and neck tumours treated with radiotherapy and healthy subjects. Reverse-phase high-pressure liquid chromatography was used for protein separation. Nine protein fractions (including acidic and basic proline-rich proteins (PRPs), cystatins, histatins and statherin) were identified in saliva from irradiated patients as well as healthy subjects. However, compared with non-irradiated healthy subjects, the fraction of acidic PRPs was significantly reduced in irradiated patients. These data indicate an alteration of the relative amount of low molecular weight salivary proteins in irradiated patients besides the reduction of salivary flow.
Assuntos
Proteínas e Peptídeos Salivares/efeitos da radiação , Proteínas de Fase Aguda/efeitos da radiação , Idoso , Cromatografia Líquida de Alta Pressão , Cistatinas/efeitos da radiação , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Pessoa de Meia-Idade , Peso Molecular , Peptídeos/efeitos da radiação , Fosfoproteínas/efeitos da radiação , Prolina/efeitos da radiação , Domínios Proteicos Ricos em Prolina , Proteínas/efeitos da radiação , Saliva/metabolismo , Saliva/efeitos da radiação , Taxa Secretória/efeitos da radiaçãoRESUMO
We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (gamma-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent gamma-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced gamma-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of gamma-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects.
Assuntos
Dano ao DNA , Íons Pesados , Histonas/efeitos da radiação , Transferência Linear de Energia , Fosfoproteínas/efeitos da radiação , Efeito Espectador , Radiação Cósmica , Fibroblastos/efeitos da radiação , Imunofluorescência , Humanos , Ferro , Fosforilação , Polietileno , Doses de Radiação , Proteção Radiológica , SilícioRESUMO
DeltaNp63alpha, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, DeltaNp63alpha is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on DeltaNp63alpha posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that DeltaNp63alpha protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, DeltaNp63alpha phosphorylation was significantly modulated. Additionally, DeltaNp63alpha protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased DeltaNp63alpha ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for DeltaNp63alpha regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and DeltaNp63alpha protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.
Assuntos
Regulação da Expressão Gênica , Queratinócitos/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Ubiquitina/metabolismo , Raios Ultravioleta , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Genes Supressores de Tumor/efeitos da radiação , Genes p53/efeitos dos fármacos , Genes p53/efeitos da radiação , Humanos , Queratinócitos/efeitos da radiação , Paclitaxel/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/efeitos da radiação , Fosforilação , Complexo de Endopeptidases do Proteassoma/fisiologia , Inibidores de Proteassoma , Transativadores/genética , Transativadores/efeitos da radiação , Fatores de Transcrição , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
At any point in time, net protein phosphorylation represents the contribution of protein kinase and protein phosphatase activities affecting a specific site on a given substrate. Preservation of phosphorylated proteins in neural tissues has traditionally included flash-freezing or fresh tissue processing following tissue isolation. Rapid heat inactivation of protein kinases and phosphatases by focused microwave irradiation sacrifice represents another method to preserve, in vivo, brain protein phosphorylation state. In this study, we compared preservation of the phosphorylation state of a variety of phosphoproteins in the brain following sacrifice of mice by decapitation, decapitation into liquid nitrogen and focused microwave irradiation. We found that microwave irradiation generally provided the highest and most consistent levels of protein phosphorylation, regardless of the substrates examined in striatum and hippocampus. In general, flash-freezing resulted in the least preservation of phospho-state with ERK1/2 and CREB showing almost complete dephosphorylation. When regions of freshly decapitated brains were homogenized and incubated on ice for 30 min, ERK1/2 phosphorylation was completely lost, whereas it was well preserved in microwaved samples left at room temperature for 2 h. Loss of ERK1/2 phosphorylation in the fresh samples could not be attributed to substrate proteolysis. Our results indicate that focused microwave irradiation sacrifice may be required to achieve biologically relevant data for the in vivo protein phosphorylation state of many phosphoproteins.
Assuntos
Encéfalo/efeitos da radiação , Micro-Ondas , Fosfoproteínas/efeitos da radiação , Animais , Western Blotting/métodos , Química Encefálica , Corpo Estriado/metabolismo , Criopreservação/métodos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos da radiação , RadiaçãoRESUMO
TIMING OF CAB EXPRESSION 1 ( TOC1) functions with CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) in a transcriptional feedback loop that is important for the circadian clock in Arabidopsis thaliana (L.) Heynh. TOC1 and its four paralogues, the Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, are expressed in an intriguing daily sequence. This was proposed to form a second feedback loop, similar to the interlocking clock gene circuits in other taxa. We show that prr9 and prr5 null mutants have reciprocal period defects for multiple circadian rhythms, consistent with subtly altered expression patterns of CCA1 and TOC1. The period defects are conditional on light quality and combine additively in double-mutant plants. Thus PRR9 and PRR5 modulate light input to the circadian clock but are neither uniquely required for rhythm generation nor form a linear series of mutual PRR gene regulation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ritmo Circadiano/genética , Fatores de Transcrição/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/efeitos da radiação , DNA Bacteriano/genética , DNA de Cadeia Simples/genética , Escuridão , Luz , Mutação , Fenótipo , Fosfoproteínas/fisiologia , Fosfoproteínas/efeitos da radiação , Reação em Cadeia da Polimerase , Fatores de Transcrição/efeitos da radiaçãoRESUMO
Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.