RESUMO
Living organisms are constantly exposed to DNA damage, and optimal repair is therefore crucial. A characteristic hallmark of the response is the formation of sub-compartments around the site of damage, known as foci. Following multiple DNA breaks, the transcription factor p53 exhibits oscillations in its nuclear concentration, but how this dynamics can affect the repair remains unknown. Here, we formulate a theory for foci formation through droplet condensation and discover how oscillations in p53, with its specific periodicity and amplitude, optimize the repair process by preventing Ostwald ripening and distributing protein material in space and time. Based on the theory predictions, we reveal experimentally that the oscillatory dynamics of p53 does enhance the repair efficiency. These results connect the dynamical signaling of p53 with the microscopic repair process and create a new paradigm for the interplay of complex dynamics and phase transitions in biology.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Reparo do DNA , Dano ao DNA , Transdução de Sinais/fisiologiaRESUMO
Large-scale cell death is commonly observed during organismal development and in human pathologies1-5. These cell death events extend over great distances to eliminate large populations of cells, raising the question of how cell death can be coordinated in space and time. One mechanism that enables long-range signal transmission is trigger waves6, but how this mechanism might be used for death events in cell populations remains unclear. Here we demonstrate that ferroptosis, an iron- and lipid-peroxidation-dependent form of cell death, can propagate across human cells over long distances (≥5 mm) at constant speeds (around 5.5 µm min-1) through trigger waves of reactive oxygen species (ROS). Chemical and genetic perturbations indicate a primary role of ROS feedback loops (Fenton reaction, NADPH oxidase signalling and glutathione synthesis) in controlling the progression of ferroptotic trigger waves. We show that introducing ferroptotic stress through suppression of cystine uptake activates these ROS feedback loops, converting cellular redox systems from being monostable to being bistable and thereby priming cell populations to become bistable media over which ROS propagate. Furthermore, we demonstrate that ferroptosis and its propagation accompany the massive, yet spatially restricted, cell death events during muscle remodelling of the embryonic avian limb, substantiating its use as a tissue-sculpting strategy during embryogenesis. Our findings highlight the role of ferroptosis in coordinating global cell death events, providing a paradigm for investigating large-scale cell death in embryonic development and human pathologies.
Assuntos
Retroalimentação Fisiológica , Ferroptose , Espécies Reativas de Oxigênio , Animais , Embrião de Galinha , Humanos , Cistina/metabolismo , Retroalimentação Fisiológica/fisiologia , Ferroptose/fisiologia , Glutationa/metabolismo , Ferro/metabolismo , Peroxidação de Lipídeos , NADPH Oxidases/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Desenvolvimento Embrionário , Extremidades/embriologiaRESUMO
Cells metabolize nutrients through a complex metabolic and signaling network that governs redox homeostasis. At the core of this, redox regulatory network is a mutually inhibitory relationship between reduced glutathione and reactive oxygen species (ROS)-two opposing metabolites that are linked to upstream nutrient metabolic pathways (glucose, cysteine, and glutamine) and downstream feedback loops of signaling pathways (calcium and NADPH oxidase). We developed a nutrient-redox model of human cells to understand system-level properties of this network. Combining in silico modeling and ROS measurements in individual cells, we show that ROS dynamics follow a switch-like, all-or-none response upon glucose deprivation at a threshold that is approximately two orders of magnitude lower than its physiological concentration. We also confirm that this ROS switch can be irreversible and exhibits hysteresis, a hallmark of bistability. Our findings evidence that bistability modulates redox homeostasis in human cells and provide a general framework for quantitative investigations of redox regulation in humans.
Assuntos
Glutationa , Transdução de Sinais , Glutationa/metabolismo , Homeostase , Humanos , Oxirredução , Espécies Reativas de OxigênioRESUMO
The complexity and specificity of metazoan transcription are determined by combinatorial control of the composition and activity of regulatory complexes. To investigate the basis of this specificity, we focused on the glucocorticoid receptor (GR), a single regulatory factor that integrates multiple signals to give rise to many distinct patterns of expression. We measured the expression of a set of genes, each directly GR-regulated, but by different mechanisms in two cell lines. We varied ligand (dose, chemistry, and duration of treatment), GR (expression level and functionality), and a non-GR regulatory factor that commonly interacts with GR. Our study revealed distinct expression patterns within this set of genes, but all could be modeled by an incoherent feed-forward regulatory logic. Cellular signals, operating on GR and other factors within regulatory complexes, may define and modulate the kinetics and strength of the activating or inhibitory paths of the regulatory logic. Thus, characterizing systems behavior by perturbing single or multiple signals can reveal general principles of regulation, providing an approach to the dissection and deconvolution of combinatorial control.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais/genética , Linhagem Celular Tumoral , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Cinética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
An optically tunable and detectable magnetoeletric (ME) effect has been discovered in the composite consisting of InGaN/GaN multiple quantum wells and magnetostrictive ferromagnetic Ni or FeCo thin films at room temperature. Due to the interactively optical and piezoelectric properties of nitride semiconductors, this composite provides an intriguing optically accessible system, in which the magnetoelectric effect can be both easily tuned and detected. The underlying mechanism can be well accounted for by the interplay among magnetostrictive, piezoelectric and optical transition. It thus offers a new paradigm to generate artificial material systems with magnetic/electric/optical inter-related/controllable properties.
RESUMO
Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we proposed that functional buffering can be mediated via expression of buffering genes in specific cells and tissues, by which we named Cell-specific Expression-BUffering (CEBU). We developed an inference index (C-score) for CEBU by computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq. We report that C-score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results suggest CEBU as a potential buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.
Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica , Homeostase , Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de ÓrgãosRESUMO
The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.
Assuntos
Dano ao DNA/fisiologia , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteoma/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The DNA damage checkpoint kinase Rad53 is important for the survival of budding yeast under genotoxic stresses. We performed a biochemical screen to identify proteins with specific affinity for the two Forkhead associated (FHA) domains of Rad53. The N-terminal FHA1 domain was found to coordinate a complex protein interaction network, which includes nuclear proteins involved in DNA damage checkpoints and transcriptional regulation. Unexpectedly, cytosolic proteins involved in cytokinesis, including septins, were also found as FHA1 binding proteins. Consistent with this interaction, a Rad53 mutant defective in its nuclear localization was found to localize to the bud neck. Abnormal morphology was observed in cells overexpressing the FHA1 domain and in rad53Delta cells under DNA replication stress. Further, septin Shs1 appears to have an important role in the response to DNA replication stress. Collectively, the results suggest a novel function of Rad53 in the regulation of polarized cell growth in response to DNA replication stress.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Replicação do DNA , Fatores de Transcrição Forkhead/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Crescimento Celular , Polaridade Celular , Quinase do Ponto de Checagem 2 , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
This study demonstrates that chlorophosphonazo III (CPZ III) can be used as a contrast agent for photoacoustic calcium imaging. CPZ III can pass across the plasma membrane for labeling intracellular Ca2+ without cytotoxicity. In optical-resolution photoacoustic microscopy (OR-PAM), the photoacoustic (PA) signal intensity was strongly correlated with the presence of CPZ III and Ca2+ at various concentrations. The sensitivity of PA signal reception was enhanced by using an 8 MHz single-element focused ultrasound detector due to their matched frequency characteristics. Differences in the PA signal intensity were successfully found between the core and margin areas of tumorspheres in three-dimensional cell cultures. These findings indicate that CPZ III can serve as a novel PA contrast agent for functional Ca2+ imaging using OR-PAM.
RESUMO
The tumor-suppressive transcription factor p53 is a master regulator of stress responses. In non-stressed conditions, p53 is maintained at low levels by the ubiquitin ligase Mdm2 and its binding partner Mdmx. Mdmx depletion leads to a biphasic p53 response, with an initial post-mitotic pulse followed by oscillations. The mechanism underlying this dynamical behavior is unknown. Two different roles for Mdmx have been proposed: enhancing p53 ubiquitination by Mdm2 and inhibiting p53 activity. Here, we developed a mathematical model of the p53/Mdm2/Mdmx network to investigate which Mdmx functions quantitatively affect specific features of p53 dynamics under various conditions. We found that enhancement of Mdm2 activity was the most critical role of Mdmx under unstressed conditions. The model also accurately predicted p53 dynamics in Mdmx-depleted cells following DNA damage. This work outlines a strategy for rapidly testing possible network interactions to reveal those most impactful in regulating the dynamics of key proteins.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , Humanos , Células MCF-7 , Modelos Teóricos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , UbiquitinaçãoRESUMO
PURPOSE: Soft and hard tissue volumes are critical for implant placement and long-term stability. Although the literature has adequately addressed tissue biotypes of Western populations, pertinent information about Asian populations is limited. This study aimed to evaluate the soft and hard tissue profiles of the maxillary anterior teeth of the Taiwanese population using a semi-automatic algorithm. MATERIALS AND METHODS: Cone beam computed tomography images of 11 adults with well-aligned maxillary anterior teeth were overlaid with those of cast models, based on the tooth crowns manually outlined by two independent observers. Each tooth was digitally trisected mesiodistally and apicocoronally. The thicknesses of the labial gingiva and alveolar bone were measured using a customized software program. RESULTS: No obvious difference between the observers was noted regarding the dimension of tooth crowns. The average thicknesses of the labial gingiva, the labial alveolar bone, and the palatal alveolar bone were 1.76 ± 0.11 mm, 1.02 ± 0.12 mm, and 1.80 ± 0.31 mm, respectively, with no significant differences between teeth. All parameters were thicker in the apical region than in the cervical region, and the alveolar bone was thinner in the midlabial region of incisors than in the interproximal regions. The thinnest areas were the midcervical compartment of the right central incisor (0.53 ± 0.33 mm) for the labial gingiva, the midcervical compartment of the right lateral incisor (0.23 ± 0.10 mm) for the labial alveolar bone, and the mesiocervical compartment of the left central incisor (0.33 ± 0.09 mm) for the palatal alveolar bone. CONCLUSION: This study presents an objective and comprehensive methodology for evaluating the soft and hard tissue profiles of maxillary anterior teeth and may be of value for presurgical planning for immediate implant placement. The results suggest that profiles of the Taiwanese subjects are similar to profiles of Western populations.
Assuntos
Processo Alveolar/anatomia & histologia , Implantes Dentários , Gengiva/anatomia & histologia , Maxila/anatomia & histologia , Adulto , Algoritmos , Processo Alveolar/diagnóstico por imagem , Tomografia Computadorizada de Feixe Cônico/métodos , Feminino , Gengiva/diagnóstico por imagem , Humanos , Masculino , Maxila/diagnóstico por imagem , Pessoa de Meia-Idade , Palato , Software , Coroa do DenteRESUMO
BACKGROUND: This study evaluates the impact of an increase in cigarette tax in Taiwan in terms of the effects it has on the overall economy and the health benefits that it brings. METHODS: The multisector computable general equilibrium (CGE) model was used to simulate the impact of reduced cigarette consumption resulting from a new tax scheme on the entire economy gains and on health benefits. RESULTS: The results predict that because of the new tax scheme, there should be a marked reduction in cigarette consumption but a notable increase in health benefits that include saving between 28,125 and 56,250 lives. This could save NTD 1.222 approximately 2.445 billion (where USD 1 = NTD 34.6) annually in life-threatening, cigarette-related health insurance expenses which exceeds the projected decrease of NTD 1.275 billion in Gross Domestic Product (GDP) because of reduced consumption and therefore tax revenue. CONCLUSION: Overall, the increased cigarette excise tax will be beneficial in terms of both the health of the general public and the economy as a whole.
Assuntos
Modelos Econômicos , Saúde Ocupacional , Saúde Pública/tendências , Fumar/economia , Seguridade Social/tendências , Impostos/legislação & jurisprudência , Indústria do Tabaco/legislação & jurisprudência , Adulto , Efeitos Psicossociais da Doença , Análise Custo-Benefício , Eficiência , Custos de Cuidados de Saúde , Humanos , Reembolso de Seguro de Saúde , Pessoa de Meia-Idade , Saúde Pública/economia , Anos de Vida Ajustados por Qualidade de Vida , Fumar/efeitos adversos , Fumar/epidemiologia , Seguridade Social/economia , Taiwan/epidemiologia , Indústria do Tabaco/economiaRESUMO
Drug combination is an appealing strategy for combating the heterogeneity of tumors and evolution of drug resistance. However, the rationale underlying combinatorial therapy is often not well established due to lack of understandings of the specific pathways responding to the drugs, and their temporal dynamics following each treatment. Here we present several emerging trends in harnessing properties of biological systems for the optimal design of drug combinations, including the type of drugs, specific concentration, sequence of addition and the temporal schedule of treatments. We highlight recent studies showing different approaches for efficient design of drug combinations including single-cell signaling dynamics, adaption and pathway crosstalk. Finally, we discuss novel and feasible approaches that can facilitate the optimal design of combinatorial therapy.
Assuntos
Combinação de Medicamentos , Desenho de Fármacos , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
The oncogene MDMX is overexpressed in many cancers, leading to suppression of the tumor suppressor p53. Inhibitors of the oncogene product MDMX therefore might help reactivate p53 and enhance the efficacy of DNA-damaging drugs. However, we currently lack a quantitative understanding of how MDMX inhibition affects the p53 signaling pathway and cell sensitivity to DNA damage. Live cell imaging showed that MDMX depletion triggered two distinct phases of p53 accumulation in single cells: an initial postmitotic pulse, followed by low-amplitude oscillations. The response to DNA damage was sharply different in these two phases; in the first phase, MDMX depletion was synergistic with DNA damage in causing cell death, whereas in the second phase, depletion of MDMX inhibited cell death. Thus a quantitative understanding of signal dynamics and cellular states is important for designing an optimal schedule of dual-drug administration.
Assuntos
Antineoplásicos/administração & dosagem , Dano ao DNA , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Imagem Molecular , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: This study uses cigarette price elasticity to evaluate the effect of a new excise tax increase on cigarette consumption and to investigate responses from various types of smokers. METHODS: Our sample consisted of current smokers between 17 and 69 years old interviewed during an annual face-to-face survey conducted by Taiwan National Health Research Institutes between 2000 to 2003. We used Ordinary Least Squares (OLS) procedure to estimate double logarithmic function of cigarette demand and cigarette price elasticity. RESULTS: In 2002, after Taiwan had enacted the new tax scheme, cigarette price elasticity in Taiwan was found to be -0.5274. The new tax scheme brought about an average annual 13.27 packs/person (10.5%) reduction in cigarette consumption. Using the cigarette price elasticity estimate from -0.309 in 2003, we calculated that if the Health and Welfare Tax were increased by another NT 3 dollars per pack and cigarette producers shifted this increase to the consumers, cigarette consumption would be reduced by 2.47 packs/person (2.2%). The value of the estimated cigarette price elasticity is smaller than one, meaning that the tax will not only reduce cigarette consumption but it will also generate additional tax revenues. Male smokers who had no income or who smoked light cigarettes were found to be more responsive to changes in cigarette price. CONCLUSIONS: An additional tax added to the cost of cigarettes would bring about a reduction in cigarette consumption and increased tax revenues. It would also help reduce incidents smoking-related illnesses. The additional tax revenues generated by the tax increase could be used to offset the current financial deficiency of Taiwan's National Health Insurance program and provide better public services.
Assuntos
Fumar/economia , Fumar/epidemiologia , Impostos/legislação & jurisprudência , Indústria do Tabaco/economia , Adolescente , Adulto , Idoso , Comportamento do Consumidor/economia , Custos e Análise de Custo , Feminino , Necessidades e Demandas de Serviços de Saúde/economia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Econométricos , Programas Nacionais de Saúde , Abandono do Hábito de Fumar , Prevenção do Hábito de Fumar , Taiwan/epidemiologia , Indústria do Tabaco/legislação & jurisprudênciaRESUMO
Assembly of SAS-6 dimers to form the centriolar cartwheel requires the ZYG-1/Plk4 kinase. Here, we show that ZYG-1 recruits SAS-6 to the mother centriole independently of its kinase activity; kinase activity is subsequently required for cartwheel assembly. We identify a direct interaction between ZYG-1 and the SAS-6 coiled coil that explains its kinase activity-independent function in SAS-6 recruitment. Perturbing this interaction, or the interaction between an adjacent segment of the SAS-6 coiled coil and SAS-5, prevented SAS-6 recruitment and cartwheel assembly. SAS-6 mutants with alanine substitutions in a previously described ZYG-1 target site or in 37 other residues, either phosphorylated by ZYG-1 in vitro or conserved in closely related nematodes, all supported cartwheel assembly. We propose that ZYG-1 binding to the SAS-6 coiled coil recruits the SAS-6-SAS-5 complex to the mother centriole, where a ZYG-1 kinase activity-dependent step, whose target is unlikely to be SAS-6, triggers cartwheel assembly.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Centríolos/genética , Sequência Conservada , Embrião não Mamífero/metabolismo , Ativação Enzimática , Masculino , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Quinases/genética , Multimerização Proteica , Interferência de RNA , TransgenesRESUMO
Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Catálise , Proteínas de Ciclo Celular/química , Quinase do Ponto de Checagem 2 , Relação Dose-Resposta a Droga , Epitopos/química , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de AminoácidosRESUMO
Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O(2) consumption approximately 20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as gamma-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Células COS , Chlorocebus aethiops , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Interferon gama/farmacologia , Proteínas Mitocondriais/genética , Oxigênio/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Proteínas de Ligação a RNA , Ratos , Proteínas Ribossômicas/genética , Estaurosporina/farmacologiaRESUMO
Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.