RESUMO
Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.
Assuntos
Proteína Supressora de Tumor p53 , Humanos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ciclo Celular , Divisão Celular , Proteína Supressora de Tumor p53/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismoRESUMO
Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.
Assuntos
DNA Helicases , DNA Primase , Replicação do DNA , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA , Proteína Homóloga a MRE11 , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Primase/metabolismo , DNA Primase/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Dano ao DNA , Ftalazinas/farmacologia , Piperazinas/farmacologia , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)2(5,5'dmb)]2+ and [Ru(PIP)2(5,5'dmb)]2+ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5'dmb)]2+ acts to block DNA replication fork progression.
Assuntos
Complexos de Coordenação , Rutênio , Rutênio/farmacologia , Rutênio/química , Transferência Ressonante de Energia de Fluorescência , DNA/química , Sítios de Ligação , Complexos de Coordenação/farmacologia , Complexos de Coordenação/químicaRESUMO
Cyclin-dependent kinases (CDKs) coordinate cell cycle checkpoints with DNA repair mechanisms that together maintain genome stability. However, the myriad mechanisms that can give rise to genome instability are still to be fully elucidated. Here, we identify CDK18 (PCTAIRE 3) as a novel regulator of genome stability, and show that depletion of CDK18 causes an increase in endogenous DNA damage and chromosomal abnormalities. CDK18-depleted cells accumulate in early S-phase, exhibiting retarded replication fork kinetics and reduced ATR kinase signaling in response to replication stress. Mechanistically, CDK18 interacts with RAD9, RAD17 and TOPBP1, and CDK18-deficiency results in a decrease in both RAD17 and RAD9 chromatin retention in response to replication stress. Importantly, we demonstrate that these phenotypes are rescued by exogenous CDK18 in a kinase-dependent manner. Collectively, these data reveal a rate-limiting role for CDK18 in replication stress signalling and establish it as a novel regulator of genome integrity.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Instabilidade Genômica , Transdução de Sinais , Estresse Fisiológico , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Aberrações Cromossômicas , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Dano ao DNA , Humanos , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Genome stability is maintained by a number of elegant mechanisms, which sense and repair damaged DNA. Germline defects that compromise genomic integrity result in cancer predisposition, exemplified by rare syndromes caused by mutations in certain DNA repair genes. These individuals often exhibit other symptoms including progeria and neurodegeneration. Paradoxically, some of these deleterious genetic alterations provide novel therapeutic opportunities to target cancer cells; an excellent example of such an approach being the recent development of poly (ADP-ribose) polymerase inhibitors as the first 'synthetic lethal' medicine for patients with BRCA-mutant cancers. The therapeutic exploitation of synthetic lethal interactions has enabled a novel approach to personalised medicine based on continued molecular profiling of patient and tumour material. This profiling may also aid clinicians in the identification of specific drug resistance mechanisms following relapse, and enable appropriate modification of the therapeutic regimen. This chapter focuses on therapeutic strategies designed to target aspects of the DNA damage response, and examines emerging themes demonstrating mechanistic overlap between DNA repair and neurodegeneration.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Medicina de Precisão , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genéticaRESUMO
Here, we identify coiled-coil domain-containing protein 13 (Ccdc13) in a genome-wide RNA interference screen for regulators of genome stability. We establish that Ccdc13 is a newly identified centriolar satellite protein that interacts with PCM1, Cep290 and pericentrin and prevents the accumulation of DNA damage during mitotic transit. Depletion of Ccdc13 results in the loss of microtubule organisation in a manner similar to PCM1 and Cep290 depletion, although Ccdc13 is not required for satellite integrity. We show that microtubule regrowth is enhanced in Ccdc13-depleted cells, but slowed in cells that overexpress Ccdc13. Furthermore, in serum-starved cells, Ccdc13 localises to the basal body, is required for primary cilia formation and promotes the localisation of the ciliopathy protein BBS4 to both centriolar satellites and cilia. These data highlight the emerging link between DNA damage response factors, centriolar and peri-centriolar satellites and cilia-associated proteins and implicate Ccdc13 as a centriolar satellite protein that functions to promote both genome stability and cilia formation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Centríolos/metabolismo , Cílios/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , TransfecçãoRESUMO
Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/ß-cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology, we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalized skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families, the father of the proband was found to be a mosaic for the MYH7 mutation. In eight cases, de novo mutation appeared to have occurred, which was proven in four. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalized floppiness, and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in nine of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases.
Assuntos
Miosinas Cardíacas/genética , Miopatias Distais/diagnóstico , Miopatias Distais/genética , Mutação , Cadeias Pesadas de Miosina/genética , Fenótipo , Adolescente , Adulto , Idoso , Biópsia , Miosinas Cardíacas/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Cadeias Pesadas de Miosina/metabolismo , Adulto JovemRESUMO
The centrosome acts as a centre for microtubule organisation and plays crucial roles in cell polarity, migration, growth and division. Cep131 has recently been described as a basal body component essential for cilium formation, but its function in non-ciliogenic cells is unknown. We identified human Cep131 (also known as AZI1) in a screen for regulators of genome stability. We show that centrosomal localisation of Cep131 is cell-cycle-regulated and requires both an intact microtubule network and a functional dynein-dynactin transport system. Cep131 is recruited to centriolar satellites by PCM1, and localised to the centriolar core region by both pericentrin and Cep290. Depletion of Cep131 results in a reduction in proliferation rate, centriole amplification, an increased frequency of multipolar mitosis, chromosomal instability and an increase in post-mitotic DNA damage. These data therefore highlight the importance of human Cep131 for maintaining genomic integrity.
Assuntos
Proteínas de Ciclo Celular , Centríolos , Centrossomo , Instabilidade Genômica , Proteínas dos Microtúbulos , Antígenos de Neoplasias/metabolismo , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centríolos/genética , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Instabilidade Cromossômica , Proteínas do Citoesqueleto , Complexo Dinactina , Dineínas/metabolismo , Humanos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/genética , Proteínas de Neoplasias/metabolismoRESUMO
MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site (55)RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Humanos , Camundongos , Fosforilação , Proteínas Recombinantes/química , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
MAPK phosphatase-1 (DUSP1/MKP-1) is a mitogen and stress-inducible dual specificity protein phosphatase, which can inactivate all three major classes of MAPK in mammalian cells. DUSP1/MKP-1 is implicated in cellular protection against a variety of genotoxic insults including hydrogen peroxide, ionizing radiation, and cisplatin, but its role in the interplay between different MAPK pathways in determining cell death and survival is not fully understood. We have used pharmacological and genetic tools to demonstrate that DUSP1/MKP-1 is an essential non-redundant regulator of UV-induced cell death in mouse embryo fibroblasts (MEFs). The induction of DUSP1/MKP-1 mRNA and protein in response to UV radiation is mediated by activation of the p38alpha but not the JNK1 or JNK2 MAPK pathways. Furthermore, we identify MSK1 and -2 and their downstream effectors cAMP-response element-binding protein/ATF1 as mediators of UV-induced p38alpha-dependent DUSP1/MKP-1 transcription. Dusp1/Mkp-1 null MEFs display increased signaling through both the p38alpha and JNK MAPK pathways and are acutely sensitive to UV-induced apoptosis. This lethality is rescued by the reintroduction of wild-type DUSP1/MKP-1 and by a mutant of DUSP1/MKP-1, which is unable to bind to either p38alpha or ERK1/2, but retains full activity toward JNK. Importantly, whereas small interfering RNA-mediated knockdown of DUSP1/MKP-1 sensitizes wild-type MEFs to UV radiation, DUSP1/MKP-1 knockdown in MEFS lacking JNK1 and -2 does not result in increased cell death. Our results demonstrate that cross-talk between the p38alpha and JNK pathways mediated by induction of DUSP1/MKP-1 regulates the cellular response to UV radiation.
Assuntos
Fosfatase 1 de Especificidade Dupla/genética , Fibroblastos/efeitos da radiação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Tolerância a Radiação , Animais , Morte Celular/efeitos da radiação , Células Cultivadas , Fosfatase 1 de Especificidade Dupla/análise , Fosfatase 1 de Especificidade Dupla/efeitos da radiação , Camundongos , RNA Mensageiro/análise , Tolerância a Radiação/genética , Receptor Cross-Talk , Transdução de Sinais , Ativação Transcricional/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Myasthenia gravis is an autoimmune disorder of the neuromuscular junction. Rituximab (RTX), a monoclonal antibody to CD20, leads to B lymphocyte depletion and has been used in some autoimmune disorders, including small case series of myasthenia gravis patients. METHODS: A retrospective analysis was performed of all patients with acetylcholine receptor (AChR) (11 subjects) or muscle specific kinase antibody (MuSK) positive myasthenia gravis (three subjects), who had been treated with RTX in Brisbane, Australia. In most patients 1 g of RTX, in two divided doses, was given. Patients were monitored by serial clinical assessments, flow cytometry of peripheral blood B lymphocytes and antibody testing. RESULTS: RTX led to a significant improvement in symptoms in 11 of 14 patients. Doses of immunosuppressive medications were able to be reduced in 12 of 14 patients but medications could be completely ceased in only one patient. A demonstrable reduction of autoantibody levels was found in only three AChR positive patients and one MuSK positive patient, independent of clinical improvement. Peripheral blood B lymphocyte depletion was achieved in 13 out of 14 patients. B lymphocyte recovery occurred between 9 and 30 months post RTX (median 12.3 months) and was consistently associated with worsening of clinical symptoms. CONCLUSION: Rituximab at a dose of 1 g appears to be beneficial in the treatment of patients with severe myasthenia gravis. Serial monitoring of peripheral blood B lymphocytes appears to be useful in guiding the need for further RTX therapy.
Assuntos
Anticorpos Monoclonais Murinos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Monitoramento de Medicamentos/métodos , Fatores Imunológicos/uso terapêutico , Miastenia Gravis/tratamento farmacológico , Adulto , Idoso , Autoanticorpos/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Miastenia Gravis/diagnóstico , Miastenia Gravis/imunologia , Estudos Retrospectivos , RituximabRESUMO
DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling.
Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-ets/metabolismo , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Fosfatase 6 de Especificidade Dupla/genética , Ativação Enzimática , Retroalimentação Fisiológica/fisiologia , Regulação da Expressão Gênica , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Alinhamento de Sequência , TransgenesRESUMO
It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.
Assuntos
Bornaviridae/metabolismo , Ciclo Celular/genética , Instabilidade Genômica , Microtúbulos/metabolismo , Nucleoproteínas/metabolismo , Proteína Quinase CDC2 , Linhagem Celular , Centrossomo/metabolismo , Ciclina B1/metabolismo , Dano ao DNA , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Nucleoproteínas/deficiência , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response.
Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Endodesoxirribonucleases , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/efeitos da radiação , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos da radiaçãoRESUMO
The mechanisms by which trophic factors bring about spinal motor neuron (MN) survival and regulate their number during development are not well understood. We have developed an organotypic slice culture model for the in vitro study of the trophic requirements and cell death pathways in MNs of postnatal day 1-2 mice. Both lateral motor column (LMC) and medial motor column (MMC) neurons died within 72 hr when grown in serum-free medium without trophic factors. Brain-derived neurotrophic factor, ciliary neurotrophic factor, and 8-(4-chlorophenylthio)-cAMP promoted the survival of a proportion of the neurons, but glial cell line-derived neurotrophic factor (GDNF) was the most effective trophic factor, supporting approximately 60% of MNs for 1 week in culture. Homozygous deficiency for bax, a proapoptotic member of the Bcl-2 family, saved the same proportion of neurons as GDNF, suggesting that GDNF alone was sufficient to maintain all "rescuable" MNs for at least 1 week. Analysis of MN survival in GFRalpha-1(-/-) mice demonstrated that the trophic effect of GDNF was completely mediated by its preferred coreceptor, GDNF family receptor alpha-1 (GFRalpha-1). None of the other GDNF family ligands supported significant MN survival, suggesting that there is little ligand-coreceptor cross talk within the slice preparation. Although MN subtypes can be clearly defined by both anatomical distribution and ontogenetic specification, the pattern of trophic factor responsiveness of neurons from the MMC was indistinguishable from that seen in the LMC. Thus, in contrast to all other factors and drugs studied to date, GDNF is likely to be a critical trophic agent for all early postnatal MN populations.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/análogos & derivados , Proteínas de Drosophila , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Medula Espinal/fisiologia , Animais , Animais Recém-Nascidos , Apoptose , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Sobrevivência Celular/fisiologia , Fator Neurotrófico Ciliar/farmacologia , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Homozigoto , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neurônios Motores/citologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/metabolismo , Medula Espinal/citologia , Tionucleotídeos/farmacologia , Proteína X Associada a bcl-2RESUMO
The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).
RESUMO
We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders.
Assuntos
Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Antígenos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/metabolismo , Quinase 1 do Ponto de Checagem , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Mitomicina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas de Ligação a Telômeros/antagonistas & inibidores , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We report the first Australian families with inclusion-body myopathy, Paget's disease of the bone and frontotemporal dementia (IBMPFD). The clinical characteristics of the two pedigrees are described including a previously undescribed phenotypic feature of pyramidal tract dysfunction in one family member. A novel mutation in the valosin-containing protein (VCP) gene (p.Arg155Leu) was found in one family while the other family had a previously reported mutation (p.Leu198Trp). Our findings broaden the phenotypic spectrum of IBMPFD and further emphasise the resemblance to amyotrophic lateral sclerosis in some cases.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Saúde da Família , Demência Frontotemporal/genética , Corpos de Inclusão/patologia , Doenças Musculares/genética , Osteíte Deformante/genética , Adulto , Fosfatase Alcalina/sangue , Arginina/genética , Austrália , Creatina Quinase/sangue , Eletrodiagnóstico/métodos , Feminino , Demência Frontotemporal/complicações , Humanos , Leucina/genética , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Doenças Musculares/complicações , Mutação/genética , Osteíte Deformante/complicações , Triptofano/genética , Proteína com ValosinaRESUMO
DUSP5 is an inducible, nuclear, dual-specificity phosphatase, which specifically interacts with and inactivates the ERK1/2 MAP kinases in mammalian cells. In addition, expression of DUSP5 causes nuclear translocation of ERK2 indicating that it may act as a nuclear anchor for the inactive kinase. Here we show that induction of DUSP5 mRNA and protein in response to growth factors is dependent on ERK1/2 activation and that the accumulation of DUSP5 protein is regulated by rapid proteasomal degradation. DUSP5 is phosphorylated by ERK1/2 both in vitro and in vivo on three sites (Thr321, Ser346 and Ser376) within its C-terminal domain. DUSP5 phosphorylation is absolutely dependent on the conserved kinase interaction motif (KIM) within the amino-terminal domain of DUSP5, indicating that the same protein-protein contacts are required for both the inactivation of ERK2 by DUSP5 and for DUSP5 to act as a substrate for this MAPK. Using a combination of pharmacological inhibitors and phospho-site mutants we can find no evidence that phosphorylation of DUSP5 by ERK2 significantly affects either the half-life of the DUSP5 protein or its ability to bind to, inactivate or anchor ERK2 in the nucleus. However, co-expression of ERK2 results in significant stabilisation of DUSP5, which is accompanied by reduced levels of DUSP5 ubiquitination. These changes are independent of ERK2 kinase activity but absolutely depend on the ability of ERK2 to bind to DUSP5. We conclude that DUSP5 is stabilised by complex formation with its physiological substrate and that this may reinforce its activity as both a phosphatase and nuclear anchor for ERK2.