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1.
Mol Cells ; 42(7): 530-545, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31362469

RESUMO

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.


Assuntos
Carcinoma Hepatocelular/genética , Quimiocina CXCL12/genética , Histonas/metabolismo , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Microambiente Tumoral/genética , Regulação para Cima/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiocina CXCL12/metabolismo , Epigênese Genética/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Compostos Heterocíclicos/farmacologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacologia , Transcrição Genética/efeitos da radiação , Microambiente Tumoral/efeitos da radiação , Raios X
2.
Oncogene ; 38(4): 549-563, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30158672

RESUMO

Ionizing radiation (IR) is a conventional cancer therapeutic, to which cancer cells develop radioresistance with exposure. The residual cancer cells after radiation treatment also have increased metastatic potential. The mechanisms by which cancer cells develop radioresistance and gain metastatic potential are still unknown. In this study acute IR exposure induced cancer cell senescence and apoptosis, but after long-term IR exposure, cancer cells exhibited radioresistance. The proliferation of radioresistant cells was retarded, and most cells were arrested in G0/G1 phase. The radioresistant cells simultaneously showed resistance to further IR-induced apoptosis, premature senescence, and epithelial to mesenchymal transformation (EMT). Acute IR exposure steadily elevated CDC6 protein levels due to the attenuation of ubiquitination, while CDC6 overexpression was observed in the radioresistant cells because the insufficiency of CDC6 phosphorylation blocked protein translocation from nucleus to cytoplasm, resulting in subcellular protein accumulation when the cells were arrested in G0/G1 phase. CDC6 ectopic overexpression in CNE2 cells resulted in apoptosis resistance, G0/G1 cell cycle arrest, premature senescence, and EMT, similar to the characteristics of radioresistant CNE2-R cells. Targeting CDC6 with siRNA promoted IR-induced senescence, sensitized cancer cells to IR-induced apoptosis, and reversed EMT. Furthermore, CDC6 depletion synergistically repressed the growth of CNE2-R xenografts when combined with IR. The study describes for the first time cell models for IR-induced senescence, apoptosis resistance, and EMT, three major mechanisms by which radioresistance develops. CDC6 is a novel radioresistance switch regulating senescence, apoptosis, and EMT. These studies suggest that CDC6highKI67low represents a new diagnostic marker of radiosensitivity, and CDC6 represents a new therapeutic target for cancer radiosensitization.


Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Apoptose/efeitos da radiação , Carcinoma/patologia , Senescência Celular/fisiologia , Transição Epitelial-Mesenquimal/efeitos da radiação , Neoplasias Nasofaríngeas/patologia , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional/efeitos da radiação , Tolerância a Radiação/fisiologia , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Carcinoma/radioterapia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Antígeno Ki-67/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/radioterapia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação/efeitos da radiação , Estabilidade Proteica , Transporte Proteico/efeitos da radiação , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitinação/efeitos da radiação , Raios X
3.
Food Funct ; 9(9): 4936-4947, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30178790

RESUMO

Syzygium aromaticum L., commonly named clove, is widely used in the food industry due to its antioxidant and antibacterial capabilities. However, little information is available regarding its role in resisting skin photoaging. This study investigated 50% ethanol extract of Syzygium aromaticum L. (SA) and eugenol (EO) for anti-aging effects in UVB-irradiated normal human dermal fibroblasts (NHDFs) and hairless mice. In vitro, SA and EO suppressed matrix metalloproteinase-1, 3 (MMP-1 and MMP-3) secretion as well as the activator protein 1 (AP-1) phosphorylation. SA and EO also activated nuclear erythroid 2-related factor/antioxidant-response element (Nrf2/ARE) signaling which improves the antioxidant activity and inhibited nuclear factor-κB (NF-κB) and interleukin-6 (IL-6) expression, pro-inflammatory factors. Furthermore, SA and EO suppressed the nuclear factor of activated T cells c1 (NFATc1) which is a known activator of MMPs, cooperator transforming growth factor beta (TGF-ß) and NF-κB in Ca2+/calcineurin-regulated transcription. In vivo, SA significantly improved the levels of procollagen type I and elastin through TGF/Smad signaling. The histopathological studies found that SA reduced wrinkles. SA also increased filament aggregating protein (filaggrin), which repairs the skin barrier function and improved the skin's hydration. Altogether, SA effectively ameliorated UVB-induced photoaging. It is expected to become a promising natural product.


Assuntos
Suplementos Nutricionais , Topos Floridos/química , Extratos Vegetais/uso terapêutico , Lesões Experimentais por Radiação/terapia , Pele/efeitos da radiação , Syzygium/química , Cicatrização , Animais , Antioxidantes/uso terapêutico , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Eugenol/uso terapêutico , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Camundongos Pelados , Óleos Voláteis/uso terapêutico , Estresse Oxidativo/efeitos da radiação , Fosforilação/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Lesões Experimentais por Radiação/imunologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Distribuição Aleatória , Pele/imunologia , Pele/metabolismo , Pele/patologia , Envelhecimento da Pele/imunologia , Envelhecimento da Pele/patologia , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos
4.
Arch Physiol Biochem ; 124(2): 185-193, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28906145

RESUMO

This study was designed to evaluate the effect of rutin on PI3K/AKT-signalling in case of acrylamide or γ-radiation-induced neurotoxicity. To induce brain damage, animals were received acrylamide (25 mg/kg b.wt./orally/day) or 5 Gy of γ-radiation exposure accompanied with an administration of rutin (200 mg/kg b.wt./orally/day). Our data revealed that, compared to acrylamide or γ-radiation, rutin activated PI3K/AKT/GSK-3ß/NRF-2-pathway through increased protein levels of p-PI3K, p-AKT and p-GSK-3ß and up-regulated the expression of NRF-2. This was achieved by modulating MDA, GST, IL-1ß, IL-6 and reduced the interference of ROS with IGF-1 and NGF stimulating the PI3K/AKT-signaling. Furthermore, histopathological examinations of brain tissues showed that rutin has modulated tissue architecture after acrylamide or γ-radiation induced tissue damage. It could be concluded that rutin provides protection effect against acrylamide or γ-radiation-induced neurotoxicity via activation of the PI3K/AKT/GSK-3ß/NRF-2-pathway by altering the phosphorylation state through its ability to scavenge free radicals generation, modulating gene expression and its anti-inflammatory effects.


Assuntos
Acrilamida/toxicidade , Raios gama/efeitos adversos , Fármacos Neuroprotetores/uso terapêutico , Síndromes Neurotóxicas/prevenção & controle , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/uso terapêutico , Rutina/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Antioxidantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos da radiação , Suplementos Nutricionais , Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Masculino , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos da radiação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/efeitos da radiação , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Lesões por Radiação/imunologia , Lesões por Radiação/metabolismo , Lesões por Radiação/patologia , Ratos Sprague-Dawley
5.
J Radiat Res ; 59(1): 18-26, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29040655

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disease leading to progressive loss of memory and other cognitive functions. One of the well-known pathological markers of AD is the accumulation of amyloid-beta protein (Aß), and its plaques, in the brain. Recent studies using Tg-5XFAD mice as a model of AD have reported that exposure to radiofrequency electromagnetic fields (RF-EMF) from cellular phones reduced Aß plaques in the brain and showed beneficial effects on AD. In this study, we examined whether exposure to 1950 MHz RF-EMF affects Aß processing in neural cells. We exposed HT22 mouse hippocampal neuronal cells and SH-SY5Y human neuroblastoma cells to RF-EMF (SAR 6 W/kg) for 2 h per day for 3 days, and analyzed the mRNA and protein expression of the key genes related to Aß processing. When exposed to RF-EMF, mRNA levels of APP, BACE1, ADAM10 and PSEN1 were decreased in HT22, but the mRNA level of APP was not changed in SH-SY5Y cells. The protein expression of APP and BACE1, as well as the secreted Aß peptide, was not significantly different between RF-EMF-exposed 7w-PSML, HT22 and SH-SY5Y cells and the unexposed controls. These observations suggest that RF-EMF exposure may not have a significant physiological effect on Aß processing of neural cells in the short term. However, considering that we only exposed HT22 and SH-SY5Y cells to RF-EMF for 2 h per day for 3 days, we cannot exclude the possibility that 1950 MHz RF-EMF induces physiological change in Aß processing with long-term and continuous exposure.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Campos Eletromagnéticos , Hipocampo/citologia , Neuroblastoma/metabolismo , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Ondas de Rádio , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Neurônios/efeitos da radiação
6.
PLoS One ; 12(12): e0188535, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29200431

RESUMO

Increased interest in clinical application of photodynamic therapy (PDT) in various medical fields poses a demand for better understanding of processes triggered by photo-treatment. Most of the work on PDT performed so far has focused on the immediate effects of photo-treatment. It is generally accepted that cellular damage occurs during light exposure and within a short period thereafter. If cells are not killed during the PDT, they might recover, depending on the extent of the photo-induced damage. Little is known, however, about the relationship between the properties of photosensitizers (PSs) and the delayed consequences of PDT. The aim of this work was to investigate cellular responses to sub-lethal photodynamic treatment and how toxicogenic potency may be affected by molecular features of the PS. Results demonstrated that for cationic porphyrin-based PSs, lipophilicity is the main factor determining the fate of the cells in the 24-hour post-illumination period. PSs with amphiphilic properties initiated oxidative reactions that continued in the dark, long after light exposure, and caused suppression of metabolism and loss of cell viability with concomitant changes in electrophoretic mobility of proteins, including caspases. Apoptotic activity was not stimulated in the post-illumination period. This study demonstrated that in PDT mediated by amphiphilic cationic metalloporphyrin PSs, even when immediate photo-damage is relatively mild, destructive oxidative processes initiated during PDT continue in the absence of light to substantially impair metabolism, and that post-illumination protein modification may modify utilization of cell death pathways.


Assuntos
Morte Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Luz/efeitos adversos , Metaloporfirinas/efeitos adversos , Fotoquimioterapia/efeitos adversos , Fármacos Fotossensibilizantes/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Metaloporfirinas/química , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Fármacos Fotossensibilizantes/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Tensoativos/efeitos adversos , Tensoativos/química
7.
J Biol Chem ; 292(33): 13843-13852, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28663371

RESUMO

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática/efeitos da radiação , Estabilidade Enzimática/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Luz , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação/efeitos da radiação , Processos Fotoquímicos , Fototropismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/efeitos da radiação , Mutação Puntual , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes de Fusão/metabolismo
8.
Anal Chem ; 89(16): 8304-8310, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28708386

RESUMO

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.


Assuntos
Cisteína/análogos & derivados , Peptídeos/química , Cisteína/química , Cisteína/efeitos da radiação , Raios Infravermelhos , Espectrometria de Massas/métodos , Oxirredução , Peptídeos/metabolismo , Peptídeos/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação
9.
J Biol Chem ; 292(37): 15321-15328, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28747438

RESUMO

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.


Assuntos
Lipoilação , Microdomínios da Membrana/metabolismo , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Rana catesbeiana/fisiologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Cisteína/química , Cistina/química , Adaptação à Escuridão , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Cinética , Luz , Lipoilação/efeitos da radiação , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos da radiação , Oxirredução , Conformação Proteica/efeitos da radiação , Multimerização Proteica/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Rodopsina/química , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/efeitos da radiação , Transducina/química , Transducina/metabolismo
10.
Autophagy ; 13(8): 1318-1330, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28594263

RESUMO

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.


Assuntos
Autofagia , Biocatálise , Proteínas Fúngicas/metabolismo , Magnaporthe/citologia , Magnaporthe/metabolismo , Processos Fototróficos , Acetilação , Autofagia/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Luz , Magnaporthe/genética , Magnaporthe/efeitos da radiação , Processos Fototróficos/efeitos da radiação , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos da radiação , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação , Transcrição Genética/efeitos da radiação
11.
Plant Cell Environ ; 40(11): 2457-2468, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27943362

RESUMO

The red/far-red light absorbing photoreceptors phytochromes regulate development and growth and thus play an essential role in optimizing adaptation of the sessile plants to the ever-changing environment. Our understanding of how absorption of a red/far-red photon by phytochromes initiates/modifies diverse physiological responses has been steadily improving. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. We discuss how its light-independent inactivation (termed dark/thermal reversion), post-translational modification, including ubiquitination, phosphorylation and sumoylation, as well as heterodimerization with other phytochrome species modify red light-controlled physiological responses. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.


Assuntos
Luz , Desenvolvimento Vegetal/efeitos da radiação , Fitocromo/química , Fitocromo/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Temperatura Ambiente
12.
Amino Acids ; 48(12): 2855-2866, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27600614

RESUMO

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, ß-, and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500 Gy) and extracted the water-soluble (WS) and water-insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation sites of asparagine and glutamine, and isomerization of aspartyl in rat α- and ß-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 and 5 Gy; however, isomerization did not occur in any crystallin even after exposure to 500 Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts.


Assuntos
Catarata/genética , Processamento de Proteína Pós-Traducional/genética , alfa-Cristalinas/metabolismo , beta-Cristalinas/metabolismo , Sequência de Aminoácidos/efeitos da radiação , Animais , Catarata/metabolismo , Eletroforese em Gel Bidimensional , Raios gama , Humanos , Cristalino/metabolismo , Cristalino/patologia , Cristalino/efeitos da radiação , Oxirredução/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Ratos , Espectrometria de Massas em Tandem , alfa-Cristalinas/química , beta-Cristalinas/química
13.
Sci Rep ; 6: 30212, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27452117

RESUMO

Lysine acetylation and succinylation are major types of protein acylation that are important in many cellular processes including gene transcription, cellular metabolism, DNA damage response. Malfunctions in these post-translational modifications are associated with genome instability and disease in higher organisms. In this study, we used high-resolution nano liquid chromatography-tandem mass spectrometry combined with affinity purification to quantify the dynamic changes of protein acetylation and succinylation in response to ultraviolet (UV)-induced cell stress. A total of 3345 acetylation sites in 1440 proteins and 567 succinylation sites in 246 proteins were identified, many of which have not been reported previously. Bioinformatics analysis revealed that these proteins are involved in many important biological processes, including cell signalling transduction, protein localization and cell metabolism. Crosstalk analysis between these two modifications indicated that modification switches might regulate protein function in response to UV-induced DNA damage. We further illustrated that FEN1 acetylation at different sites could lead to different cellular phenotypes, suggesting the multiple function involvement of FEN1 acetylation under DNA damage stress. These systematic analyses provided valuable resources and new insight into the potential role of lysine acetylation and succinylation under physiological and pathological conditions.


Assuntos
Acetilação/efeitos da radiação , Lisina/metabolismo , Estresse Fisiológico/efeitos da radiação , Ácido Succínico/metabolismo , Raios Ultravioleta/efeitos adversos , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Instabilidade Genômica/efeitos da radiação , Células HeLa , Humanos , Processamento de Proteína Pós-Traducional/efeitos da radiação
14.
Clin Cancer Res ; 22(17): 4428-39, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27076628

RESUMO

PURPOSE: Ionizing radiation (IR) induces intracellular signaling processes as part of a treatment-induced stress response. Here we investigate IR-induced ADAM17 activation and the role of ADAM17-shed factors for radiation resistance in non-small cell lung cancer. EXPERIMENTAL DESIGN: Large-scale secretome profiling was performed using antibody arrays. Secretion kinetics of ADAM17 substrates was determined using ELISA across multiple in vitro and in vivo models of non-small cell lung cancer. Clonogenic survival and tumor xenograft assays were performed to determine radiosensitization by ADAM17 inhibition. RESULTS: On the basis of a large-scale secretome screening, we investigated secretion of auto- or paracrine factors in non-small cell lung cancer in response to irradiation and discovered the ADAM17 network as a crucial mediator of resistance to IR. Irradiation induced a dose-dependent increase of furin-mediated cleavage of the ADAM17 proform to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB signaling in otherwise cetuximab-resistant target cells, and enhanced IR-induced cytotoxicity. The combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in vivo demonstrating the potential of ADAM17 targeting in combination with radiotherapy. CONCLUSIONS: Radiotherapy activates ADAM17 in non-small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and IR-induced treatment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of non-small cell lung cancer. Clin Cancer Res; 22(17); 4428-39. ©2016 AACR.


Assuntos
Proteína ADAM17/metabolismo , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteoma , Proteômica , Tolerância a Radiação , Proteína ADAM17/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Ativação Enzimática/efeitos da radiação , Furina/metabolismo , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteômica/métodos , Interferência de RNA , Tolerância a Radiação/genética , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Oncol Rep ; 35(5): 3101-5, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26986008

RESUMO

The COP9/signalosome (CSN) multi-protein complex regulates the activity of cullin-RING ubiquitin ligases (CRLs), including the DDB2 and CSA CRL4 ligases (CRL4DDB2 and CRL4CSA), which are involved in the repair of UV-induced DNA damages. In the present study, we demonstrated that the protein kinase ATM, a key component of the DNA damage response (DDR), phosphorylates CSN1 and CSN7a, two subunits of the CSN complex, in a UV-dependent manner. The phosphorylation of CSN1 on serine 474 was detected as early as 3 h after UV-exposure, peaked at 8 h and persisted until 48 h post-UV irradiation. Such a time course suggests a role in late DDR rather than in DNA repair. Consistently, overexpression of a phosphorylation-resistant S474A CSN1 mutant reduced UV-induced apoptosis. Thus, CSN1 appears to play a role not only in DNA repair but also in UV-induced apoptosis.


Assuntos
Apoptose/efeitos da radiação , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Raios Ultravioleta , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Complexo do Signalossomo COP9 , Dano ao DNA , Reparo do DNA , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação
16.
Mol Biosyst ; 12(4): 1388-93, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26940144

RESUMO

Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells.


Assuntos
Inteínas , Luz , Processamento de Proteína Pós-Traducional/efeitos da radiação , Animais , Linhagem Celular , Escherichia coli , Humanos , Modelos Moleculares , Conformação Proteica/efeitos da radiação , Domínios Proteicos , Processamento de Proteína/efeitos da radiação
17.
PLoS One ; 11(2): e0150175, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26918332

RESUMO

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.


Assuntos
Histonas/efeitos da radiação , Queratinócitos/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Luz Solar , Raios Ultravioleta , Acetilação/efeitos da radiação , Divisão Celular , Linhagem Celular Transformada , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica/efeitos da radiação , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/efeitos da radiação , Histona Desacetilases/metabolismo , Histona Desacetilases/efeitos da radiação , Histonas/metabolismo , Humanos , Queratinócitos/metabolismo , Lisina/metabolismo
18.
Mol Cell ; 61(3): 419-433, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26774286

RESUMO

FBXW7 is a haploinsufficient tumor suppressor with loss-of-function mutations occurring in human cancers. FBXW7 inactivation causes genomic instability, but the mechanism remains elusive. Here we show that FBXW7 facilitates nonhomologous end-joining (NHEJ) repair and that FBXW7 depletion causes radiosensitization. In response to ionizing radiation, ATM phosphorylates FBXW7 at serine 26 to recruit it to DNA double-strand break (DSB) sites, whereas activated DNA-PKcs phosphorylates XRCC4 at serines 325/326, which promotes binding of XRCC4 to FBXW7. SCF(FBXW7) E3 ligase then promotes polyubiquitylation of XRCC4 at lysine 296 via lysine 63 linkage for enhanced association with the Ku70/80 complex to facilitate NHEJ repair. Consistent with these findings, a small-molecule inhibitor that abrogates XRCC4 polyubiquitylation reduces NHEJ repair. Our study demonstrates one mechanism by which FBXW7 contributes to genome integrity and implies that inactivated FBXW7 in human cancers could be a strategy for increasing the efficacy of radiotherapy.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/metabolismo , Neoplasias Pancreáticas/enzimologia , Poliubiquitina/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Ciclopentanos/farmacologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Células HCT116 , Humanos , Lisina , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/radioterapia , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Pirimidinas/farmacologia , Interferência de RNA , Tolerância a Radiação , Radiossensibilizantes/farmacologia , Fatores de Tempo , Transfecção , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/antagonistas & inibidores , Ubiquitinas/metabolismo
19.
DNA Cell Biol ; 35(3): 140-5, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26717101

RESUMO

The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Raios Ultravioleta/efeitos adversos , Citoplasma/metabolismo , Citoplasma/efeitos da radiação , Citoesqueleto/metabolismo , Enzima Desubiquitinante CYLD , Células HeLa/efeitos da radiação , Humanos , Células MCF-7/efeitos da radiação , Microtúbulos/metabolismo , Microtúbulos/efeitos da radiação , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transporte Proteico/efeitos da radiação
20.
Int J Mol Sci ; 16(12): 29996-30014, 2015 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-26694365

RESUMO

Histone H2AX plays a crucial role in molecular and cellular responses to DNA damage and in the maintenance of genome stability. It is downstream of ataxia telangiectasia mutated (ATM) damage signaling pathway and there is an emerging role of the transcription factor FoxO3a, a regulator of a variety of other pathways, in activating this signaling. We asked whether H2AX may feedback to FoxO3a to affect respective FoxO3a-dependent pathways. We used a genetically matched pair of mouse embryonic fibroblast H2AX(+/+) and H2AX(-/-) cell lines to carry out comprehensive time-course and dose-response experiments and to show that the expression of several FoxO3a-regulated genes was altered in H2AX(-)(/-) compared to H2AX(+/+) cells at both basal and irradiated conditions. Hspa1b and Gadd45a were down-regulated four- to five-fold and Ddit3, Cdkn1a and Sod2 were up-regulated 2-3-fold in H2AX(-/-) cells. Using the luciferase reporter assay, we directly demonstrated that transcriptional activity of FoxoO3a was reduced in H2AX(-/-) cells. FoxO3a localization within the nuclear phospho-ATM (Ser1981) foci in irradiated cells was affected by the H2AX status, as well as its posttranslational modification (phospho-Thr32). These differences were associated with genomic instability and radiosensitivity in H2AX(-/-) cells. Finally, knockdown of H2AX in H2AX(+/+) cells resulted in FoxO3a-dependent gene expression patterns and increased radiosensitivity that partially mimicked those found in H2AX(-/-) cells. Taken together, our data suggest a role for FoxO3a in the maintenance of genome integrity in response to DNA damage that is mediated by H2AX via yet unknown mechanisms.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Instabilidade Genômica/efeitos da radiação , Histonas/metabolismo , Radiação Ionizante , Transcrição Genética/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Proteína Forkhead Box O3 , Regulação da Expressão Gênica/efeitos da radiação , Técnicas de Silenciamento de Genes , Histonas/deficiência , Espaço Intracelular/metabolismo , Espaço Intracelular/efeitos da radiação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Transporte Proteico/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Reprodutibilidade dos Testes , Fatores de Tempo
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