RESUMO
Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break ("nick") adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts.
Assuntos
Clivagem do DNA , Animais , Ácido Apurínico/genética , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Humanos , Hidrólise , Oligonucleotídeos/química , Oligonucleotídeos/genética , Estresse OxidativoRESUMO
The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24h, whereas MDM2 levels declined after 6h exposure, but rose over the next 18h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Biotinilação , Linhagem Celular , Precipitação Química , DNA/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Técnicas de Silenciamento de Genes , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/deficiência , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Camundongos , Dados de Sequência Molecular , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Estreptavidina/metabolismo , UbiquitinaçãoRESUMO
Human ribosomal protein S3 (RPS3) has previously been shown to have alternative roles beyond its participation in protein synthesis. For example, our in vitro studies have shown that RPS3 has an extraordinarily high binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG). Notably, in cells exposed to oxidative stress RPS3 translocates to the nucleus where it co-localizes with foci of 8-oxoG. We have engineered transgenic mice over expressing RPS3 in an attempt to determine the outcome of RPS3 translocation in a whole animal. Mouse embryonic fibroblasts (MEFs) isolated from these transgenic mice showed an increased accumulation of DNA damage in cells exposed to oxidative damage when compared to MEFs from wild-type mice. In MEFs exposed to oxidative stress we observed the translocation of RPS3 from the cytoplasm to the nucleus and co-localizing to 8-oxoG foci, an observation that could involve the blocking of the repair of this mutagenic base and thereby explain why transgenic MEFs exposed to oxidative stress have higher levels of DNA damage.
Assuntos
Reparo do DNA , Proteínas Ribossômicas/metabolismo , Animais , Dano ao DNA , Desoxiguanosina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Regulação para CimaRESUMO
NF-kappaB is a DNA-binding protein complex that transduces a variety of activating signals from the cytoplasm to specific sets of target genes. To understand the preferential recruitment of NF-kappaB to specific gene regulatory sites, we used NF-kappaB p65 in a tandem affinity purification and mass spectrometry proteomic screen. We identified ribosomal protein S3 (RPS3), a KH domain protein, as a non-Rel subunit of p65 homodimer and p65-p50 heterodimer DNA-binding complexes that synergistically enhances DNA binding. RPS3 knockdown impaired NF-kappaB-mediated transcription of selected p65 target genes but not nuclear shuttling or global protein translation. Rather, lymphocyte-activating stimuli caused nuclear translocation of RPS3, parallel to p65, to form part of NF-kappaB bound to specific regulatory sites in chromatin. Thus, RPS3 is an essential but previously unknown subunit of NF-kappaB involved in the regulation of key genes in rapid cellular activation responses. Our observations provide insight into how NF-kappaB selectively controls gene expression.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/metabolismo , Humanos , Células Jurkat , Dados de Sequência Molecular , Complexos Multiproteicos/química , NF-kappa B/química , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: The aging process occurs at variable rates both among and within species and may be related to the variability in oxygen consumption and free radical production impacting oxidative stress. The current study was designed to test whether nonagenarians have a relatively low metabolic rate and whether it is associated with low levels of oxidative stress relative to age. METHODS: Resting metabolic rate (RMR) and markers of oxidative stress to lipids, proteins, and DNA were measured in three groups of individuals aged 20-34 (n=47), 60-74 (n=49), and>or=90 years (n=74). RESULTS: RMR, adjusted for fat-free mass, fat mass, and sex, was lower in both older groups when compared to the young group (pAssuntos
Envelhecimento/fisiologia
, Metabolismo Basal/fisiologia
, Estresse Oxidativo/fisiologia
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Composição Corporal
, DNA/metabolismo
, Feminino
, Humanos
, Metabolismo dos Lipídeos
, Masculino
, Pessoa de Meia-Idade
, Proteínas/metabolismo
RESUMO
Besides its role in translation and ribosome maturation, human ribosomal protein S3 (hS3) is implicated in DNA damage recognition as reflected by its affinity for abasic sites and 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA in vitro. Here, we demonstrate that hS3 is capable of carrying out both roles by its ex vivo translocation from the cytoplasm to the nucleus as a consequence of genotoxic stress. The translocation of hS3 is dependent on ERK1/2-mediated phosphorylation of a threonine residue (T42) of hS3. Two different ectopically expressed site-directed mutants of T42 failed to respond to conditions of genotoxic stress, thus providing a link between DNA damage and ERK1/2 dependent phosphorylation of hS3. Lastly, hS3 was traced in exposed cells to its co-localization with 8-oxoG foci, raising the possibility that hS3 is a member of a cellular DNA damage response pathway that results in its interaction with sites of DNA damage.
Assuntos
Dano ao DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mutagênicos/toxicidade , Proteínas Ribossômicas/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Transporte Proteico , Proteínas Ribossômicas/genéticaRESUMO
BACKGROUND: Caloric restriction without malnutrition extends life span in a range of organisms including insects and mammals and lowers free radical production by the mitochondria. However, the mechanism responsible for this adaptation are poorly understood. METHODS AND FINDINGS: The current study was undertaken to examine muscle mitochondrial bioenergetics in response to caloric restriction alone or in combination with exercise in 36 young (36.8 +/- 1.0 y), overweight (body mass index, 27.8 +/- 0.7 kg/m(2)) individuals randomized into one of three groups for a 6-mo intervention: Control, 100% of energy requirements; CR, 25% caloric restriction; and CREX, caloric restriction with exercise (CREX), 12.5% CR + 12.5% increased energy expenditure (EE). In the controls, 24-h EE was unchanged, but in CR and CREX it was significantly reduced from baseline even after adjustment for the loss of metabolic mass (CR, -135 +/- 42 kcal/d, p = 0.002 and CREX, -117 +/- 52 kcal/d, p = 0.008). Participants in the CR and CREX groups had increased expression of genes encoding proteins involved in mitochondrial function such as PPARGC1A, TFAM, eNOS, SIRT1, and PARL (all, p < 0.05). In parallel, mitochondrial DNA content increased by 35% +/- 5% in the CR group (p = 0.005) and 21% +/- 4% in the CREX group (p < 0.004), with no change in the control group (2% +/- 2%). However, the activity of key mitochondrial enzymes of the TCA (tricarboxylic acid) cycle (citrate synthase), beta-oxidation (beta-hydroxyacyl-CoA dehydrogenase), and electron transport chain (cytochrome C oxidase II) was unchanged. DNA damage was reduced from baseline in the CR (-0.56 +/- 0.11 arbitrary units, p = 0.003) and CREX (-0.45 +/- 0.12 arbitrary units, p = 0.011), but not in the controls. In primary cultures of human myotubes, a nitric oxide donor (mimicking eNOS signaling) induced mitochondrial biogenesis but failed to induce SIRT1 protein expression, suggesting that additional factors may regulate SIRT1 content during CR. CONCLUSIONS: The observed increase in muscle mitochondrial DNA in association with a decrease in whole body oxygen consumption and DNA damage suggests that caloric restriction improves mitochondrial function in young non-obese adults.
Assuntos
Restrição Calórica , Exercício Físico/fisiologia , Mitocôndrias Musculares/fisiologia , Sobrepeso/fisiologia , Adiponectina/fisiologia , Adulto , Dano ao DNA/fisiologia , Metabolismo Energético/fisiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Potencial da Membrana Mitocondrial , Pessoa de Meia-Idade , Músculo Esquelético/ultraestrutura , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Sirtuína 1 , Sirtuínas/biossínteseRESUMO
Human ribosomal protein S3 (hS3) has a high apparent binding affinity for the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG). The hS3 ribosomal protein has also been found to inhibit the base excision repair (BER) enzyme hOGG1 from liberating 8-oxoG residing in a 5'-end-labeled oligonucleotide. To understand the in vivo involvement of hS3 in BER, we have turned to RNA interference to generate knockdown of hS3 in cells exposed to DNA damaging agents. Here we show that a 40% knockdown of hS3 resulted in as much as a seven-fold increase in the 24h survival-rate of HEK293 cells exposed to hydrogen peroxide. Significant protection to the alkylating agent methyl methanesulfonate (MMS) was also observed. Protection to the chemotherapeutic alkylating agent Thio-TEPA was only revealed at longer exposure times where the agent became more toxic to untransfected human cells. Overall, these results raise the possibility that hS3 interferes with the repair of the DNA lesions produced by genotoxic agents that potentially could play a role in the onset of cancer and other pathological states such as aging.
Assuntos
Dano ao DNA/genética , Proteínas Ribossômicas/antagonistas & inibidores , Alquilantes/farmacologia , Antineoplásicos Alquilantes/farmacologia , Western Blotting , Sobrevivência Celular , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Humanos , Peróxido de Hidrogênio/farmacologia , Metanossulfonato de Metila/farmacologia , Oxidantes/farmacologia , Plasmídeos , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.
Assuntos
Guanosina/análogos & derivados , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , DNA/química , DNA/genética , DNA/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Guanosina/metabolismo , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break ("nick") adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts.
Assuntos
Ácido Apurínico/análise , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA/análise , Polinucleotídeos/análise , Álcalis/química , Animais , Eletroforese , Humanos , Marcação por Isótopo , Desnaturação de Ácido Nucleico , Oligonucleotídeos/química , Uracila/químicaRESUMO
CONTEXT: Prolonged calorie restriction increases life span in rodents. Whether prolonged calorie restriction affects biomarkers of longevity or markers of oxidative stress, or reduces metabolic rate beyond that expected from reduced metabolic mass, has not been investigated in humans. OBJECTIVE: To examine the effects of 6 months of calorie restriction, with or without exercise, in overweight, nonobese (body mass index, 25 to <30) men and women. DESIGN, SETTING, AND PARTICIPANTS: Randomized controlled trial of healthy, sedentary men and women (N = 48) conducted between March 2002 and August 2004 at a research center in Baton Rouge, La. INTERVENTION: Participants were randomized to 1 of 4 groups for 6 months: control (weight maintenance diet); calorie restriction (25% calorie restriction of baseline energy requirements); calorie restriction with exercise (12.5% calorie restriction plus 12.5% increase in energy expenditure by structured exercise); very low-calorie diet (890 kcal/d until 15% weight reduction, followed by a weight maintenance diet). MAIN OUTCOME MEASURES: Body composition; dehydroepiandrosterone sulfate (DHEAS), glucose, and insulin levels; protein carbonyls; DNA damage; 24-hour energy expenditure; and core body temperature. RESULTS: Mean (SEM) weight change at 6 months in the 4 groups was as follows: controls, -1.0% (1.1%); calorie restriction, -10.4% (0.9%); calorie restriction with exercise, -10.0% (0.8%); and very low-calorie diet, -13.9% (0.7%). At 6 months, fasting insulin levels were significantly reduced from baseline in the intervention groups (all P<.01), whereas DHEAS and glucose levels were unchanged. Core body temperature was reduced in the calorie restriction and calorie restriction with exercise groups (both P<.05). After adjustment for changes in body composition, sedentary 24-hour energy expenditure was unchanged in controls, but decreased in the calorie restriction (-135 kcal/d [42 kcal/d]), calorie restriction with exercise (-117 kcal/d [52 kcal/d]), and very low-calorie diet (-125 kcal/d [35 kcal/d]) groups (all P<.008). These "metabolic adaptations" (~ 6% more than expected based on loss of metabolic mass) were statistically different from controls (P<.05). Protein carbonyl concentrations were not changed from baseline to month 6 in any group, whereas DNA damage was also reduced from baseline in all intervention groups (P <.005). CONCLUSIONS: Our findings suggest that 2 biomarkers of longevity (fasting insulin level and body temperature) are decreased by prolonged calorie restriction in humans and support the theory that metabolic rate is reduced beyond the level expected from reduced metabolic body mass. Studies of longer duration are required to determine if calorie restriction attenuates the aging process in humans. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00099151.
Assuntos
Restrição Calórica , Longevidade/fisiologia , Sobrepeso/fisiologia , Estresse Oxidativo/fisiologia , Adulto , Composição Corporal , Índice de Massa Corporal , Temperatura Corporal , Dano ao DNA , Fragmentação do DNA , Sulfato de Desidroepiandrosterona/metabolismo , Metabolismo Energético , Exercício Físico , Feminino , Glucose/metabolismo , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , DescansoRESUMO
Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break ("nick") adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Animais , Ácido Apurínico/análise , DNA/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Drosophila/química , Proteínas de Escherichia coli/química , Furanos/química , Oligodesoxirribonucleotídeos/química , Polinucleotídeos/análiseRESUMO
The human ribosomal protein S3 (hS3) possesses associated activities that suggest alternative roles beyond its participation in protein translation. For example, it is capable of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction, an activity that is missing in partially purified extracts of xeroderma pigmentosum group-D fibroblasts. In a recent study, we showed by surface plasmon resonance (SPR) that hS3 also has a very high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) and AP sites in DNA. Using the same SPR technology, it is shown here that hS3 positively interacts with the human base excision repair (BER) enzymes N-glycosylase/AP lyase OGG1 and APE/Ref-1. Using a DNA substrate that allows for the detection of 8-oxoG repair, we also show that hOGG1 N-glycosylase activity becomes increasingly more robust in the presence of hS3. Human S3 was found to co-immunoprecipitate with both hOGG1 and APE/Ref-1, indicating that these proteins physically interact with one another. These results raise the possibility that hS3 not only functions as a ribosomal protein but, in addition, may influence repair activities at sites of DNA damage.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Guanosina/análogos & derivados , Proteínas Ribossômicas/metabolismo , DNA Bacteriano/metabolismo , Guanosina/metabolismo , Humanos , Imunoprecipitação , Oligonucleotídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Ressonância de Plasmônio de Superfície/métodosRESUMO
The human ribosomal protein S3 (hS3) possesses multifunctional activities that are involved in both protein translation, as well as the ability of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction. We recently showed that hS3 also has a surprising binding affinity for an 7,8-dihydro-8-oxoguanine (8-oxoG) residue embedded in a 5' end labeled 37mer DNA oligonucleotide. To understand the interaction of hS3 and DNA templates containing 8-oxoG, we carried out real-time analysis using surface plasmon resonance (SPR). Notably, hS3 was found to have an apparent three orders of magnitude higher binding affinity (KD) for 8-oxoG than the human N-glycosylase/AP lyase base excision repair (BER) enzyme OGG1. An even more dramatic five orders of magnitude higher binding affinity for AP DNA was found for hS3 as opposed to hOGG1. These results suggest that ribosomal protein hS3 may have a multifunctional role that may also affect functions associated with DNA base excision repair transactions.