RESUMO
Self-rated health (SRH) is one of the most frequently used indicators in health and social research. Its robust association with mortality in very different populations implies that it is a comprehensive measure of health status and may even reflect the condition of the human organism beyond clinical diagnoses. Yet the biological basis of SRH is poorly understood. We used data from three independent European population samples (N approx. 15,000) to investigate the associations of SRH with 150 biomolecules in blood or urine (biomarkers). Altogether 57 biomarkers representing different organ systems were associated with SRH. In almost half of the cases the association was independent of disease and physical functioning. Biomarkers weakened but did not remove the association between SRH and mortality. We propose three potential pathways through which biomarkers may be incorporated into an individual's subjective health assessment, including (1) their role in clinical diseases; (2) their association with health-related lifestyles; and (3) their potential to stimulate physical sensations through interoceptive mechanisms. Our findings indicate that SRH has a solid biological basis and it is a valid but non-specific indicator of the biological condition of the human organism.
Assuntos
Biomarcadores , Autoavaliação Diagnóstica , Nível de Saúde , Autorrelato , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Biomarcadores/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The paper describes the importance of toxicology as a discipline, its past achievements, current scientific challenges, and future development. Toxicological expertise is instrumental in the reduction of human health risks arising from chemicals and drugs. Toxicological assessment is needed to evaluate evidence and arguments, whether or not there is a scientific base for concern. The immense success already achieved by toxicological work is exemplified by reduced pollution of air, soil, water, and safer working places. Predominantly predictive toxicological testing is derived from the findings to assess risks to humans and the environment. Assessment of the adversity of molecular effects (including epigenetic effects), the effects of mixtures, and integration of exposure and biokinetics into in vitro testing are emerging challenges for toxicology. Toxicology is a translational science with its base in fundamental science. Academic institutions play an essential part by providing scientific innovation and education of young scientists.
Assuntos
Medição de Risco/métodos , Testes de Toxicidade/métodos , Toxicologia/organização & administração , Animais , Exposição Ambiental/efeitos adversos , Exposição Ambiental/prevenção & controle , Poluentes Ambientais/toxicidade , Alemanha , Humanos , Sociedades Científicas , Toxicologia/métodosRESUMO
MARK-AGE is a recently completed European population study, where bioanalytical and anthropometric data were collected from human subjects at a large scale. To facilitate data analysis and mathematical modelling, an extended database had to be constructed, integrating the data sources that were part of the project. This step involved checking, transformation and documentation of data. The success of downstream analysis mainly depends on the preparation and quality of the integrated data. Here, we present the pre-processing steps applied to the MARK-AGE data to ensure high quality and reliability in the MARK-AGE Extended Database. Various kinds of obstacles that arose during the project are highlighted and solutions are presented.
Assuntos
Envelhecimento/fisiologia , Bases de Dados Factuais , Armazenamento e Recuperação da Informação , Confidencialidade , Feminino , Humanos , MasculinoRESUMO
AIM: To examine associations of DNA damage, cardiovascular risk factors and physical performance with vitality, in middle-aged men. We also sought to elucidate underlying factors of physical performance by comparing physical performance parameters to DNA damage parameters and cardiovascular risk factors. METHODS: We studied 2487 participants from the Metropolit cohort of 11 532 men born in 1953 in the Copenhagen Metropolitan area. The vitality level was estimated using the SF-36 vitality scale. Cardiovascular risk factors were determined by body mass index (BMI), and haematological biochemistry tests obtained from non-fasting participants. DNA damage parameters were measured in peripheral blood mononuclear cells (PBMCs) from as many participants as possible from a representative subset of 207 participants. RESULTS: Vitality was inversely associated with spontaneous DNA breaks (measured by comet assay) (P = 0.046) and BMI (P = 0.002), and positively associated with all of the physical performance parameters (all P < 0.001). Also, we found several associations between physical performance parameters and cardiovascular risk factors. In addition, the load of short telomeres was inversely associated with maximum jump force (P = 0.018), with lowered significance after exclusion of either arthritis sufferers (P = 0.035) or smokers (P = 0.031). CONCLUSION: Here, we show that self-reported vitality is associated with DNA breaks, BMI and objective (measured) physical performance in a cohort of middle-aged men. Several other associations in this study verify clinical observations in medical practice. In addition, the load of short telomeres may be linked to peak performance in certain musculoskeletal activities.
Assuntos
Doenças Cardiovasculares/metabolismo , Dano ao DNA/genética , Exercício Físico/fisiologia , Índice de Massa Corporal , Doenças Cardiovasculares/fisiopatologia , Estudos de Coortes , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , AutoimagemRESUMO
Psychological stress has often been described as a feeling of being overwhelmed by the necessity of constant adjustment to an individual's changing environment. Stress affects people of all ages, but the lives of the elderly may particularly be affected. Major changes can cause anxiety leading to feelings of insecurity and/or loss of self-esteem and depression. The cellular mechanisms underlying psychological stress are poorly understood. This review focuses on the physical and molecular consequences of psychological stress linked to aging processes and, in particular, how molecular changes induced by psychological stress can compromise healthy aging.
Assuntos
Envelhecimento/fisiologia , Estresse Psicológico/fisiopatologia , Fármacos do Sistema Nervoso Central/uso terapêutico , Doença Crônica , Dano ao DNA/fisiologia , Doenças do Sistema Endócrino/fisiopatologia , Terapia por Exercício/métodos , Humanos , Doenças do Sistema Imunitário/fisiopatologia , Doenças do Sistema Nervoso/fisiopatologia , Psicoterapia/métodos , Estresse Psicológico/terapiaRESUMO
The prevalence of age-related diseases is increased in individuals with post-traumatic stress disorder (PTSD). However, the underlying biological mechanisms are still unclear. N-glycosylation is an age-dependent process, identified as a biomarker for physiological aging (GlycoAge Test). To investigate whether traumatic stress accelerates the aging process, we analyzed the N-glycosylation profile in n=13 individuals with PTSD, n=9 trauma-exposed individuals and in n=10 low-stress control subjects. Individuals with PTSD and trauma-exposed individuals presented an upward shift in the GlycoAge Test, equivalent to an advancement of the aging process by 15 additional years. Trauma-exposed individuals presented an intermediate N-glycosylation profile positioned between severely traumatized individuals with PTSD and low-stress control subjects. In conclusion, our data suggest that cumulative exposure to traumatic stressors accelerates the process of physiological aging.
Assuntos
Senilidade Prematura/metabolismo , Polissacarídeos/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo , Estresse Psicológico/metabolismo , Adulto , Senilidade Prematura/sangue , Estudos de Casos e Controles , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Polissacarídeos/sangue , Transtornos de Estresse Pós-Traumáticos/sangue , Estresse Psicológico/sangue , Adulto JovemRESUMO
It is believed that normal cells with an unaffected DNA damage response (DDR) and DNA damage repair machinery, could be less prone to DNA damaging treatment than cancer cells. However, the anticancer drug, etoposide, which is a topoisomerase II inhibitor, can generate DNA double strand breaks affecting not only replication but also transcription and therefore can induce DNA damage in non-replicating cells. Indeed, we showed that etoposide could influence transcription and was able to activate DDR in resting human T cells by inducing phosphorylation of ATM and its substrates, H2AX and p53. This led to activation of PUMA, caspases and to apoptotic cell death. Lymphoblastoid leukemic Jurkat cells, as cycling cells, were more sensitive to etoposide considering the level of DNA damage, DDR and apoptosis. Next, we used ATM inhibitor, KU 55933, which has been shown previously to be a radio/chemo-sensitizing agent. Pretreatment of resting T cells with KU 55933 blocked phosphorylation of ATM, H2AX and p53, which, in turn, prevented PUMA expression, caspase activation and apoptosis. On the other hand, KU 55933 incremented apoptosis of Jurkat cells. However, etoposide-induced DNA damage in resting T cells was not influenced by KU 55933 as revealed by the FADU assay. Altogether our results show that KU 55933 blocks DDR and apoptosis induced by etoposide in normal resting T cells, but increased cytotoxic effect on proliferating leukemic Jurkat cells. We discuss the possible beneficial and adverse effects of drugs affecting the DDR in cancer cells that are currently in preclinical anticancer trials.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Apoptose/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Etoposídeo/toxicidade , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Células Jurkat , Morfolinas/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pironas/farmacologia , Linfócitos T/efeitos dos fármacos , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.
Assuntos
Envelhecimento/genética , Expressão Gênica , Genes , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/normas , Adulto , Idoso , Algoritmos , Humanos , Hipoxantina Fosforribosiltransferase/metabolismo , Leucócitos Mononucleares/metabolismo , Pessoa de Meia-Idade , RNA Mensageiro/genética , Padrões de Referência , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA- and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEK's autoantigenic properties and in its function as a promoter of cell survival.
Assuntos
Apoptose/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Mutagênicos/toxicidade , Proteínas Oncogênicas/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Animais , Anticorpos Monoclonais , Artrite Juvenil/imunologia , Autoanticorpos/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Células HeLa , Humanos , Células Jurkat , Camundongos , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/imunologia , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Poly(ADP-ribosyl)ation is a posttranslational modification of proteins in eukaryotic cells catalysed by a family of NAD+ ADP-ribosyl transferases, the poly(ADP-ribose) polymerases (PARPs). PARP-encoding genes now constitute a superfamily of at least 18 members encoding proteins that share homology with the catalytic domain of the founding member, PARP-1. Poly(ADP-ribose) metabolism is of central importance in a wide variety of biological processes including maintenance of genomic stability, DNA repair, transcriptional regulation, centromere function, modulation of telomere length, regulation of proteasomal protein degradation, regulation of endosomal vesicle trafficking and apoptosis. The life cycle of poly(ADP-ribose) is discussed in the following section. In addition, an overview of the genes and proteins involved in poly(ADP-ribose) metabolism and their possible cellular function is provided.
Assuntos
Centrômero/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Telômero/metabolismo , Animais , Morte Celular/fisiologia , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição GênicaRESUMO
Nickel, cadmium, cobalt, and arsenic compounds are well-known carcinogens to humans and experimental animals. Even though their DNA-damaging potentials are rather weak, they interfere with the nucleotide and base excision repair at low, noncytotoxic concentrations. For example, both water-soluble Ni(II) and particulate black NiO greatly reduced the repair of DNA adducts induced by benzo[a]pyrene, an important environmental pollutant. Furthermore, Ni(II), As(III), and Co(II) interfered with cell cycle progression and cell cycle control in response to ultraviolet C radiation. As potential molecular targets, interactions with so-called zinc finger proteins involved in DNA repair and/or DNA damage signaling were investigated. We observed an inactivation of the bacterial formamidopyrimidine-DNA glycosylase (Fpg), the mammalian xeroderma pigmentosum group A protein (XPA), and the poly(adenosine diphosphate-ribose)polymerase (PARP). Although all proteins were inhibited by Cd(II) and Cu(II), XPA and PARP but not Fpg were inhibited by Co(II) and Ni(II). As(III) deserves special attention, as it inactivated only PARP, but did so at very low concentrations starting from 10 nM. Because DNA is permanently damaged by endogenous and environmental factors, functioning processing of DNA lesions is an important prerequisite for maintaining genomic integrity; its inactivation by metal compounds may therefore constitute an important mechanism of metal-related carcinogenicity.
Assuntos
Ciclo Celular/efeitos dos fármacos , Reparo do DNA , Metais Pesados/efeitos adversos , Dedos de Zinco , Animais , Proteínas de Ligação a DNA/farmacologia , DNA-Formamidopirimidina Glicosilase , Humanos , N-Glicosil Hidrolases/farmacologia , Poli(ADP-Ribose) Polimerases/farmacologia , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
Metal ions are essential components of biological systems; nevertheless, even essential elements may have toxic or carcinogenic properties. Thus, besides As(III) and Cd(II), also Ni(II) and Co(II) have been shown previously to disturb different types of DNA repair systems at low, non-cytotoxic concentrations. Since some metals exert high affinities for SH groups, we investigated whether zinc finger structures in DNA-binding motifs of DNA repair proteins are potential targets for toxic metal ions. The bacterial formamidopyrimidine-DNA glycosylase (Fpg protein) involved in base excision repair was inhibited by Cd(II), Cu(II) and Hg(II) with increasing efficiencies, whereas Co(II), As(III), Pb(II) and Ni(II) had no effect. Furthermore, Cd(II) still disturbed enzyme function when bound to metallothionein. Strong inhibition was also observed in the presence of phenylselenyl chloride, followed by selenocystine, while selenomethionine was not inhibitory. Regarding the mammalian XPA protein involved in the recognition of DNA lesions during nucleotide excision repair, its DNA-binding capacity was diminished by Cd(II), Cu(II), Ni(II) and Co(II), while Hg(II), Pb(II) and As(III) were ineffective. Finally, the H(2)O(2)-induced activation of the poly(ADP-ribose)polymerase (PARP) involved in DNA strand break detection and apoptosis was greatly reduced by Cd(II), Co(II), Ni(II) and As(III). Similarly, the disruption of correct p53 folding and DNA binding by Cd(II), Ni(II) and Co(II) has been shown by other authors. Therefore, zinc-dependent proteins involved in DNA repair and cell-cycle control may represent sensitive targets for some toxic metals such as Cd(II), Ni(II), Co(II) and Cu(II), as well as for some selenium compounds. Relevant mechanisms of inhibition appear to be the displacement of zinc by other transition metals as well as redox reactions leading to thiol/disulfide interchange.
Assuntos
Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Metais Pesados/toxicidade , Dedos de Zinco/efeitos dos fármacos , Zinco/toxicidade , Cátions Bivalentes , Reparo do DNA/fisiologia , Humanos , N-Glicosil Hidrolases/metabolismo , Dobramento de Proteína , Proteínas de Ligação a RNA/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A , Dedos de Zinco/genéticaRESUMO
Poly(ADP-ribose) polymerase 1 (PARP-1) lies at the basis of a DNA-interacting protein family that maintains genome integrity. Here we describe the genomic organisation of rat PARP-1 gene (Adprt), refine its assignment to rat chromosome (RNO) 13q25-->q26 by FISH and compare its genomic organisation between rat, mouse and human. It appears that in human, mouse and rat Adprt consists of 23 similar-sized exons with well-conserved intron and exon borders. Adprt orthologs map to homologous chromosome regions at the termini of the q-arms of human and mouse chromosomes 1 and rat 13, with gene order being conserved between the rodents. Kimura protein distance comparison with human PARP-1 as reference revealed the bovine protein to be the least conserved with 10.3 substitutions per 100 amino acids, followed by rat (8.6) and mouse (8.4).
Assuntos
ADP Ribose Transferases/genética , Filogenia , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Mapeamento Cromossômico , Sequência Conservada , Éxons , Genoma , Humanos , Hibridização in Situ Fluorescente , Camundongos , RatosRESUMO
One of the immediate eukaryotic cellular responses to DNA breakage is the covalent post-translational modification of nuclear proteins with poly(ADP-ribose) from NAD+ as precursor, mostly catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Recently several other polypeptides have been shown to catalyse poly(ADP-ribose) formation. Poly(ADP-ribosyl)ation is involved in a variety of physiological and pathophysiological phenomena. Physiological functions include its participation in DNA-base excision repair, DNA-damage signalling, regulation of genomic stability, and regulation of transcription and proteasomal function, supporting the previously observed correlation of cellular poly(ADP-ribosyl)ation capacity with mammalian life. The pathophysiology effects are mediated through PARP-1 overactivity, which can cause cell suicide by NAD+ depletion. It is apparent that the latter effect underlies the pathogenesis of a wide range of disease states including type-1 diabetes, ischaemic infarcts in various organs, and septic or haemorrhagic shock. Therefore pharmacological modulation of poly(ADP-ribosyl)ation may prove to be an exciting option for various highly prevalent, disabling and even lethal diseases.
Assuntos
Reparo do DNA , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cisteína Endopeptidases/genética , Dano ao DNA , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/fisiopatologia , Regulação da Expressão Gênica , Humanos , Infarto/enzimologia , Infarto/fisiopatologia , Modelos Biológicos , Complexos Multienzimáticos/genética , Complexo de Endopeptidases do Proteassoma , Transcrição GênicaRESUMO
Recent experimental work from a variety of biological systems, ranging from yeast to human beings, lends increasing support to the view that stochastic damage inflicted to biological macromolecules is the driving force for the ageing process. The damage is derived from small reactive molecules, most prominently reactive oxygen intermediates (ROI), that arise during normal cellular metabolism and are associated with important if not essential cellular functions. The major classes of macromolecules at risk are proteins, lipids and DNA, but damage to DNA (both nuclear and mitochondrial) may entail particularly severe consequences. Cellular dysfunction resulting from macromolecular damage can be detected as a variety of expressions, such as genomic instability, inappropriate cell differentiation events or cell death. While for post-mitotic cell types replacement of the dead cell by another cell of the same lineage is not possible, mitotic cell types may initially replace dead cells via cell proliferation. But exhaustion of the self-renewal capacity of the respective lineage, by either replication-associated or damage-associated telomere shortening, will ultimately also lead to loss of parenchymal cell mass and functional impairment of tissues, the latter being a typical feature of ageing of tissues and organs. It has been demonstrated in various experimental systems that the rate ageing of can be retarded by lowering the production of endogenous ROI or by improving cellular anti-oxidative defences. Whether augmentation of cellular DNA repair capacity will have the same effect remains to be seen.
Assuntos
Envelhecimento/genética , Dano ao DNA/fisiologia , Envelhecimento/fisiologia , Animais , Humanos , Substâncias Macromoleculares , Espécies Reativas de Oxigênio/fisiologiaRESUMO
Ageing is highly complex, involving multiple mechanisms at different levels. Nevertheless, recent evidence suggests that several of the most important mechanisms are linked via endogenous stress-induced DNA damage caused by reactive oxygen species (ROS). Understanding how such damage contributes to age-related changes requires that we explain how these different mechanisms relate to each other and potentially interact. In this article, we review the contributions of stress-induced damage to cellular DNA through (i) the role of damage to nuclear DNA and its repair mediated via the actions of poly(ADP-ribose) polymerase-1, (ii) the role of damage to telomeric DNA and its contribution to telomere-driven cell senescence, and (iii) the role of damage to and the accumulation of mutations in mitochondrial DNA. We describe how an integrative approach to studying these mechanisms, coupled with computational modelling, may be of considerable importance in resolving some of the complexity of cellular ageing.
Assuntos
Envelhecimento/genética , Dano ao DNA , Estresse Fisiológico/genética , Animais , Ciclo Celular , Divisão Celular , Senescência Celular , Humanos , Longevidade , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerases , TelômeroRESUMO
Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation or oxidants. The resulting formation of protein-coupled poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage, probably via its contribution to DNA base-excision repair. Furthermore, based on recent results there is a role emerging for PARP-1 as a negative regulator of genomic instability in cells under genotoxic stress. Regarding possible applications for clinical cancer therapy with DNA-damaging agents, it appears that both inhibition and up-regulation of the poly(ADP-ribosyl)ation response in the malignant cells to be eradicated are promising strategies to improve the outcome of such therapy, albeit for different reasons.
Assuntos
Aberrações Cromossômicas/genética , Dano ao DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Ativação Enzimática , Genoma , HumanosRESUMO
DNA damage induced in higher eukaryotes by alkylating agents, oxidants or ionising radiation triggers the synthesis of protein-conjugated poly(ADP-ribose) catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Previously, cellular poly(ADP-ribosyl)ation capacity has been shown to correlate positively with the life span of mammalian species [Proc. Natl. Acad. Sci. USA 89 (1992) 11,759-11,763]. Here, we have tested whether this correlation results from differences in kinetic parameters of the enzymatic activity of PARP-1. We therefore compared recombinant enzymes, expressed in a baculovirus system, from rat and man as two mammalian species with extremely divergent life span. In standard activity assays performed in the presence of histones as poly(ADP-ribose) acceptors both enzymes showed saturation kinetics with [NAD(+)]. The kinetic parameters (k(cat), k(m) and k(cat)/k(m)) of the two enzymes were not significantly different. However, in assays assessing the auto-poly(ADP-ribosyl)ation reaction, both enzymes displayed second-order kinetics with respect to [PARP-1], and up to two-fold higher specific activity was observed for human versus rat PARP-1. We conclude that the correlation of poly(ADP-ribosyl)ation capacity with life span is not reflected in the kinetic parameters, but that subtle differences in primary structure of PARP-1 from mammalian species of different longevity may control the extent of the automodification reaction.
Assuntos
Longevidade/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Baculoviridae/genética , Dano ao DNA , Humanos , Cinética , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/isolamento & purificação , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade da EspécieRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) encoded by the PARP-1 gene, is a ubiquitous and abundant DNA-binding protein involved in the cellular response to various genotoxic agents. In a previous study we showed that maximal oligonucleotide-stimulated poly(ADP-ribosyl)ation was significantly higher in permeabilised lymphoblastoid cell lines from a French population of centenarians compared with controls aged 20-70 years, supporting the notion that longevity is associated with a genetically determined, high poly(ADP-ribosyl)ation capacity. Here, we describe four new genetic polymorphisms, three of which represent silent nucleotide variants (C402T, T1011C, G1215A), and one of which leads to a valine762-to-alanine exchange (T2444C). We undertook an association study between two of these polymorphisms and human longevity or poly(ADP-ribosyl)ation capacity in permeabilised lymphoblastoid cells. By analysing 648 DNA samples from a French population (324 centenarians and 324 controls) by fluorescent-allele-specific PCR, we showed the absence of any significant enrichment of any of the genotypes in the study of centenarians versus controls. Furthermore, we studied genotype distributions from individuals who had previously been tested for poly(ADP-ribosyl)ation capacity. None of the genotype combinations at any polymorphic site studied could be related to a high or low level of poly(ADP-ribosyl)ation capacity. Together, these results strongly suggest that the longevity-related differences in the poly(ADP-ribosyl)ation capacity of human lymphoblastoid cell lines cannot be explained by genetic polymorphisms in the PARP-1 coding sequence and that other mechanisms have to be considered as potential regulators of specific poly(ADP-ribosyl)ation capacity.