RESUMO
There has been a significant increase in basal cell carcinoma (BCC) incidence, the most common cancer in humans and the age of presentation with the first diagnosis of BCC has decreased in past decades. In this study, we investigated the possibility of genetic markers that can lead to earlier and closer observation of patients at high risk for development of multiple BCCs. The overall goal is to decrease the morbidity and the economic burden of diagnosis and treatment of recurring and/or advanced BCCs. Four patients with numerous BCCs, some of them exceptionally large, were included in this study. A sample of representative BCCs, normal non-sun-exposed skin and blood samples were obtained from each patient. Whole-exome sequencing of DNA was conducted on all samples, and a series of bioinformatics filtering was performed to identify potentially pathogenic sequence variants. The analysis of the data resulted in detection of oncogenic mutations in PTCH1, two of which being novel, and concurrent mutations in TP53 in BCC tumours of all four patients. Such mutations may explain the numerous and postexcision recurring nature of the BCCs of exceptionally large size observed in all these patients, and they can be suggested to serve as a genetic marker for high-risk patients for early detection, prognostication and close follow-up.
Assuntos
Carcinoma Basocelular , Neoplasias Cutâneas , Carcinogênese , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Humanos , Mutação , Recidiva Local de Neoplasia , Receptor Patched-1/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.
Assuntos
Endotélio Vascular/metabolismo , Neovascularização Patológica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Códon , Endotélio Vascular/citologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Regulação para CimaRESUMO
The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein ß subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gß subunit.
Assuntos
Motivos de Aminoácidos/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/ultraestrutura , Receptores de Hormônio Liberador da Corticotropina/ultraestrutura , Receptores de Glucagon/ultraestrutura , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Proteína Semelhante a Receptor de Calcitonina/fisiologia , Proteína Semelhante a Receptor de Calcitonina/ultraestrutura , Sinalização do Cálcio , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Terciária de Proteína , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/fisiologia , Proteína 1 Modificadora da Atividade de Receptores/ultraestrutura , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/fisiologia , Receptores Acoplados a Proteínas G , Receptores de Glucagon/metabolismo , Receptores de Glucagon/fisiologiaAssuntos
Anti-Inflamatórios/uso terapêutico , Doenças Hereditárias Autoinflamatórias/diagnóstico , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Mutação/genética , Adolescente , Exantema , Febre , Doenças Hereditárias Autoinflamatórias/tratamento farmacológico , Doenças Hereditárias Autoinflamatórias/genética , Homozigoto , Humanos , Lactente , Proteína Antagonista do Receptor de Interleucina 1/genética , Masculino , Mucosite , Linhagem , IrmãosRESUMO
Biallelic mutations in the ITK gene cause a T-cell primary immunodeficiency with Epstein-Barr virus (EBV)-lymphoproliferative disorders. We describe a novel association of a homozygous ITK mutation with ß-human papillomavirus (HPV)-positive epidermodysplasia verruciformis. Thus, loss of function in ITK can result in broad dysregulation of T-cell responses to oncogenic viruses, including ß-HPV and EBV.
Assuntos
Epidermodisplasia Verruciforme/genética , Doença de Hodgkin/etiologia , Mutação com Perda de Função , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Linfócitos T/patologia , Acitretina/uso terapêutico , Adulto , Alelos , Tratamento Farmacológico , Epidermodisplasia Verruciforme/tratamento farmacológico , Epidermodisplasia Verruciforme/imunologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Estudos de Associação Genética , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/imunologia , Homozigoto , Humanos , Ceratolíticos/uso terapêutico , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Transtornos Linfoproliferativos/virologia , Masculino , Papillomaviridae , Irmãos , Tomografia Computadorizada por Raios XAssuntos
Proteínas de Ligação ao Cálcio/genética , DNA Recombinante/genética , Epidermodisplasia Verruciforme/genética , Predisposição Genética para Doença , Mutação/genética , Infecções por Papillomavirus/diagnóstico , Adulto , Biópsia por Agulha , Comorbidade , Consanguinidade , Epidermodisplasia Verruciforme/epidemiologia , Epidermodisplasia Verruciforme/patologia , Feminino , Humanos , Imuno-Histoquímica , Irã (Geográfico) , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Linhagem , Valor Preditivo dos Testes , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medição de Risco , Índice de Gravidade de Doença , Sequenciamento do Exoma/métodosRESUMO
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.
Assuntos
Ataxina-7/metabolismo , Proteínas Mutantes/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ataxias Espinocerebelares/fisiopatologia , Visão Ocular/efeitos dos fármacos , Animais , Ataxina-7/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Peptídeos/metabolismo , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Agregados Proteicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/complicações , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/patologiaRESUMO
Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering diseases. We developed a next generation sequencing (NGS) panel covering 21 genes associated with skin fragility disorders, and it was applied to DNA from 91 probands with the diagnosis of EB. In one patient, novel homozygous mutations were disclosed in two different, unlinked EB-associated genes: EXPH5, chr11 g.108510085G > A; p.Arg1808Ter and COL17A1, chr10 g.104077423delT; p.Thr68LeufsTer106. Consequences of the COL17A1 mutation were examined by RNAseq which revealed a complex splicing pattern predicting synthesis of a truncated polypeptide (85%) or in-frame deletion of exon 4 (15% of transcripts). Transmission electron microscopy (TEM) and immunostaining revealed findings consistent with EB simplex (EBS) and junctional EB (JEB), and clinical examination revealed a complex phenotype with features of both subtypes. This case illustrates the power of next generation sequencing in identifying mutations in patients with complex EB phenotype, with implications for genotype-phenotype correlations, prenatal testing, and genetic counseling of families at risk for recurrence.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autoantígenos/genética , Epidermólise Bolhosa Simples/diagnóstico , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Juncional/diagnóstico , Epidermólise Bolhosa Juncional/genética , Homozigoto , Mutação , Colágenos não Fibrilares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alelos , Autoantígenos/metabolismo , Análise Mutacional de DNA , Estudos de Associação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Colágenos não Fibrilares/metabolismo , Linhagem , Fenótipo , Pele/metabolismo , Pele/patologia , Colágeno Tipo XVIIRESUMO
Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica , Ratos , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Glucagon/químicaRESUMO
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
Assuntos
Receptor PAR-2/química , Receptor PAR-2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Álcoois Benzílicos/química , Álcoois Benzílicos/farmacologia , Cristalografia por Raios X , Humanos , Imidazóis/química , Imidazóis/farmacologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Cinética , Ligantes , Modelos Moleculares , Receptor PAR-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Over the past decade there has been a revolution in the field of G protein-coupled receptor (GPCR) structural biology. Many years of innovative research from different areas have come together to fuel this significant change in the fortunes of this field, which for many years was characterized by the paucity of high-resolution structures. The determination to succeed has been in part due to the recognized importance of these proteins as drug targets, and although the pharmaceutical industry has been focusing on these receptors, it can be justifiably argued and demonstrated that many of the approved and commercially successful GPCR drugs can be significantly improved to increase efficacy and/or reduce undesired side effects. In addition, many validated targets in this class remain to be drugged. It is widely recognized that application of structure-based drug design approaches can help medicinal chemists a long way toward discovering better drugs. The achievement of structural biologists in providing high-resolution insight is beginning to transform drug discovery efforts, and there are a number of GPCR drugs that have been discovered by use of structural information that are in clinical development. This review aims to highlight the key developments that have brought success to GPCR structure resolution efforts and exemplify the practical application of structural information for the discovery of adenosine A2A receptor antagonists that have potential to treat multiple conditions.
Assuntos
Receptor A2A de Adenosina/efeitos dos fármacos , Animais , Cristalografia por Raios X , Descoberta de Drogas , Humanos , Inflamação/metabolismo , Camundongos , Neoplasias/metabolismo , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Doenças Respiratórias/metabolismoRESUMO
Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.
Assuntos
Receptores CCR/antagonistas & inibidores , Receptores CCR/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/genética , Sequência Conservada , Cristalografia por Raios X , Citoplasma/metabolismo , Desenho de Fármacos , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mutagênese , Receptores CCR/genética , Receptores CCR5/química , Receptores CXCR4/químicaRESUMO
The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.
Assuntos
Agonistas do Receptor A2 de Adenosina/química , Receptor A2A de Adenosina/química , Adenosina-5'-(N-etilcarboxamida)/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Receptor A2A de Adenosina/genéticaRESUMO
G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A(2A) receptor and muscarinic M(1) receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A(2A)-StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.
Assuntos
Descoberta de Drogas/métodos , Engenharia de Proteínas/métodos , Receptor A2A de Adenosina/metabolismo , Receptor Muscarínico M1/metabolismo , Sítios de Ligação , Células Cultivadas , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ensaio RadioliganteRESUMO
DNA double-strand breaks (DSBs) can be processed by the Mre11-Rad50-Nbs1 (MRN) complex, which is essential to promote ataxia telangiectasia-mutated (ATM) activation. However, the molecular mechanisms linking MRN activity to ATM are not fully understood. Here, using Xenopus laevis egg extract we show that MRN-dependent processing of DSBs leads to the accumulation of short single-stranded DNA oligonucleotides (ssDNA oligos). The MRN complex isolated from the extract containing DSBs is bound to ssDNA oligos and stimulates ATM activity. Elimination of ssDNA oligos results in rapid extinction of ATM activity. Significantly, ssDNA oligos can be isolated from human cells damaged with ionizing radiation and injection of small synthetic ssDNA oligos into undamaged cells also induces ATM activation. These results suggest that MRN-dependent generation of ssDNA oligos, which constitute a unique signal of ongoing DSB repair not encountered in normal DNA metabolism, stimulates ATM activity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Hidrolases Anidrido Ácido , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Linhagem Celular Tumoral , DNA de Cadeia Simples/metabolismo , Proteína Homóloga a MRE11 , Xenopus laevisRESUMO
It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Ciclo Celular , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Proteínas Supressoras de Tumor/químicaRESUMO
Studies of DNA repair and the maintenance of genomic integrity are essential to understanding the etiology and pathology of cancer. The availability of the complete genome sequence of Saccharomyces cerevissiae has greatly facilitated the discovery of new genes important for DNA repair.