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1.
Genes Dev ; 24(17): 1939-50, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20810650

RESUMO

In response to DNA damage, cells activate a complex signal transduction network called the DNA damage response (DDR). To enhance our current understanding of the DDR network, we performed a genome-wide RNAi screen to identify genes required for resistance to ionizing radiation (IR). Along with a number of known DDR genes, we discovered a large set of novel genes whose depletion leads to cellular sensitivity to IR. Here we describe TTI1 (Tel two-interacting protein 1) and TTI2 as highly conserved regulators of the DDR in mammals. TTI1 and TTI2 protect cells from spontaneous DNA damage, and are required for the establishment of the intra-S and G2/M checkpoints. TTI1 and TTI2 exist in multiple complexes, including a 2-MDa complex with TEL2 (telomere maintenance 2), called the Triple T complex, and phosphoinositide-3-kinase-related protein kinases (PIKKs) such as ataxia telangiectasia-mutated (ATM). The components of the TTT complex are mutually dependent on each other, and act as critical regulators of PIKK abundance and checkpoint signaling.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ets , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Genes cdc , Estudo de Associação Genômica Ampla , Humanos , Raios Infravermelhos , Peptídeos e Proteínas de Sinalização Intracelular , Chaperonas Moleculares , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Interferência de RNA
2.
Genes Dev ; 23(6): 729-39, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19261749

RESUMO

The ability to sense and respond to DNA damage is critical to maintenance of genomic stability and the prevention of cancer. In this study, we employed a genetic screen to identify a gene, NBA1 (new component of the BRCA1 A complex), that is required for resistance to ionizing radiation. The NBA1 protein localizes to sites of DNA damage and is required for G2/M checkpoint control. Proteomic analysis revealed that NBA1 is a component of the BRCA1 A complex, which also contains Brca1/Bard1, Abra1, RAP80, BRCC36, and BRE. NBA1 is required to maintain BRE and Abra1 abundance and for the recruitment of BRCA1 to sites of DNA damage. In depth bioinformatics analysis revealed that the BRCA1 A complex bears striking similarities to the 19S proteasome complex. Furthermore, we show that four members of the BRCA1-A complex possess a polyubiquitin chain-binding capability, thus forming a complex that might facilitate the deubiquitinating activity of the deubiquitination enzyme BRCC36 or the E3 ligase activity of the BRCA1/BARD1 ligase. These findings provide a new perspective from which to view the BRCA1 A complex.


Assuntos
Proteína BRCA1/fisiologia , Proteínas de Transporte/fisiologia , Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Transporte/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Reparo do DNA/fisiologia , Enzimas Desubiquitinantes , Humanos , Lasers , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitinação
3.
Proc Natl Acad Sci U S A ; 107(43): 18475-80, 2010 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-20937877

RESUMO

Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair.


Assuntos
Dano ao DNA , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Repressoras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Reparo do DNA , Células HeLa , Humanos , Técnicas In Vitro , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas do Grupo Polycomb , Proteômica , Raios Ultravioleta/efeitos adversos
4.
Proc Natl Acad Sci U S A ; 106(12): 4701-6, 2009 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-19273838

RESUMO

The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkühn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear beta-catenin-positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.


Assuntos
Divisão Celular , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Deleção de Genes , Intestino Delgado/citologia , Fosfatases cdc25/deficiência , Animais , Blastocisto/citologia , Blastocisto/enzimologia , Células Cultivadas , Cruzamentos Genéticos , Desenvolvimento Embrionário , Células Epiteliais/ultraestrutura , Feminino , Fase G1 , Fase G2 , Genótipo , Homeostase , Intestino Delgado/enzimologia , Intestino Delgado/ultraestrutura , Masculino , Camundongos , Camundongos Knockout
5.
J Med Chem ; 65(14): 9858-9872, 2022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35819182

RESUMO

CD137 (4-1BB) is a co-stimulatory receptor on immune cells and Nectin-4 is a cell adhesion molecule that is overexpressed in multiple tumor types. Using a series of poly(ethylene glycol) (PEG)-based linkers, synthetic bicyclic peptides targeting CD137 were conjugated to Bicycles targeting Nectin-4. The resulting bispecific molecules were potent CD137 agonists that require the presence of both Nectin-4-expressing tumor cells and CD137-expressing immune cells for activity. A multipronged approach was taken to optimize these Bicycle tumor-targeted immune cell agonists by exploring the impact of chemical configuration, binding affinity, and pharmacokinetics on CD137 agonism and antitumor activity. This effort resulted in the discovery of BT7480, which elicited robust CD137 agonism and maximum antitumor activity in syngeneic mouse models. A tumor-targeted approach to CD137 agonism using low-molecular-weight, short-acting molecules with high tumor penetration is a yet unexplored path in the clinic, where emerging data suggest that persistent target engagement, characteristic of biologics, may lead to suboptimal immune response.


Assuntos
Neoplasias , Animais , Moléculas de Adesão Celular , Camundongos , Nectinas , Neoplasias/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
6.
J Immunother Cancer ; 9(11)2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34725211

RESUMO

BACKGROUND: CD137 (4-1BB) is an immune costimulatory receptor with high therapeutic potential in cancer. We are creating tumor target-dependent CD137 agonists using a novel chemical approach based on fully synthetic constrained bicyclic peptide (Bicycle®) technology. Nectin-4 is overexpressed in multiple human cancers that may benefit from CD137 agonism. To this end, we have developed BT7480, a novel, first-in-class, Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA™). METHODS: Nectin-4 and CD137 co-expression analyses in primary human cancer samples was performed. Chemical conjugation of two CD137 Bicycles to a Nectin-4 Bicycle led to BT7480, which was then evaluated using a suite of in vitro and in vivo assays to characterize its pharmacology and mechanism of action. RESULTS: Transcriptional profiling revealed that Nectin-4 and CD137 were co-expressed in a variety of human cancers with high unmet need and spatial proteomic imaging found CD137-expressing immune cells were deeply penetrant within the tumor near Nectin-4-expressing cancer cells. BT7480 binds potently, specifically, and simultaneously to Nectin-4 and CD137. In co-cultures of human peripheral blood mononuclear cells and tumor cells, this co-ligation causes robust Nectin-4-dependent CD137 agonism that is more potent than an anti-CD137 antibody agonist. Treatment of immunocompetent mice bearing Nectin-4-expressing tumors with BT7480 elicited a profound reprogramming of the tumor immune microenvironment including an early and rapid myeloid cell activation that precedes T cell infiltration and upregulation of cytotoxicity-related genes. BT7480 induces complete tumor regressions and resistance to tumor re-challenge. Importantly, antitumor activity is not dependent on continuous high drug levels in the plasma since a once weekly dosing cycle provides maximum antitumor activity despite minimal drug remaining in the plasma after day 2. BT7480 appears well tolerated in both rats and non-human primates at doses far greater than those expected to be clinically relevant, including absence of the hepatic toxicity observed with non-targeted CD137 agonists. CONCLUSION: BT7480 is a highly potent Nectin-4-dependent CD137 agonist that produces complete regressions and antitumor immunity with only intermittent drug exposure in syngeneic mouse tumor models and is well tolerated in preclinical safety species. This work supports the clinical investigation of BT7480 for the treatment of cancer in humans.


Assuntos
Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Humanos , Camundongos , Neoplasias/imunologia , Ratos , Microambiente Tumoral
7.
J Immunother Cancer ; 9(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33500260

RESUMO

BACKGROUND: In contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles). METHODS: Phage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action. RESULTS: Linking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1-2 hr), yet intermittent dosing proved effective. CONCLUSION: Tumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.


Assuntos
Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/administração & dosagem , Receptor EphA2/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Células A549 , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Células HT29 , Humanos , Células Jurkat , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , Células PC-3 , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptores OX40/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Med Chem ; 60(5): 2155-2161, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28186750

RESUMO

While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.


Assuntos
Inibidores Enzimáticos/farmacologia , Zíper de Leucina , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Cristalografia por Raios X , Inibidores Enzimáticos/química
9.
J Med Chem ; 59(10): 4711-23, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27187609

RESUMO

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.


Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/normas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade
10.
Science ; 351(6278): 1208-13, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26912361

RESUMO

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Desoxiadenosinas/metabolismo , Deleção de Genes , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Purina-Núcleosídeo Fosforilase/genética , RNA Interferente Pequeno/genética , Tionucleosídeos/metabolismo
11.
Cancer Res ; 74(12): 3294-305, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24747911

RESUMO

Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis, and mitosis, offering attractive targets for anticancer treatment. Using unbiased combination screening in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors (MEKi) in KRAS-mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and antitumor activity both in vitro and in vivo than effects observed by previously reported MEKi combinations. Altogether, our results show how a novel combination of TNKS and MEK inhibitors can be highly effective in targeting KRAS-mutant cancers by suppressing a newly discovered resistance mechanism.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Proto-Oncogênicas/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Tanquirases/metabolismo , Proteínas ras/genética , Acetamidas/administração & dosagem , Aminopiridinas/administração & dosagem , Compostos de Anilina/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Cloridrato de Erlotinib , Retroalimentação Fisiológica , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras) , Pirimidinonas/administração & dosagem , Quinazolinas/administração & dosagem , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais , Sulfonamidas/administração & dosagem , Tanquirases/antagonistas & inibidores , Tiazóis/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Res ; 73(20): 6289-98, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23980093

RESUMO

Radiotherapy is one of the mainstays of anticancer treatment, but the relationship between the radiosensitivity of cancer cells and their genomic characteristics is still not well defined. Here, we report the development of a high-throughput platform for measuring radiation survival in vitro and its validation in comparison with conventional clonogenic radiation survival analysis. We combined results from this high-throughput assay with genomic parameters in cell lines from squamous cell lung carcinoma, which is standardly treated by radiotherapy, to identify parameters that predict radiation sensitivity. We showed that activation of NFE2L2, a frequent event in lung squamous cancers, confers radiation resistance. An expression-based, in silico screen nominated inhibitors of phosphoinositide 3-kinase (PI3K) as NFE2L2 antagonists. We showed that the selective PI3K inhibitor, NVP-BKM120, both decreased NRF2 protein levels and sensitized NFE2L2 or KEAP1-mutant cells to radiation. We then combined results from this high-throughput assay with single-sample gene set enrichment analysis of gene expression data. The resulting analysis identified pathways implicated in cell survival, genotoxic stress, detoxification, and innate and adaptive immunity as key correlates of radiation sensitivity. The integrative and high-throughput methods shown here for large-scale profiling of radiation survival and genomic features of solid-tumor-derived cell lines should facilitate tumor radiogenomics and the discovery of genotype-selective radiation sensitizers and protective agents.


Assuntos
Carcinoma de Células Escamosas/radioterapia , Ensaios de Triagem em Larga Escala/métodos , Neoplasias Pulmonares/radioterapia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Tolerância a Radiação/genética
13.
Science ; 332(6035): 1313-7, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21659603

RESUMO

The DNA damage response (DDR) is brought about by a protein kinase cascade that orchestrates DNA repair through transcriptional and posttranslational mechanisms. Cell cycle arrest is a hallmark of the DDR. We screened for cells that lacked damage-induced cell cycle arrest and uncovered a critical role for Fanconi anemia and homologous recombination proteins in ATR (ataxia telangiectasia and Rad3-related) signaling. Three DDR candidates, the RNA processing protein INTS7, the circadian transcription factor CLOCK, and a previously uncharacterized protein RHINO, were recruited to sites of DNA damage. RHINO independently bound the Rad9-Rad1-Hus1 complex (9-1-1) and the ATR activator TopBP1. RHINO was recruited to sites of DNA damage by the 9-1-1 complex to promote Chk1 activation. We suggest that RHINO functions together with the 9-1-1 complex and TopBP1 to fully activate ATR.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Quimiocinas/fisiologia , Reparo do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Quimiocinas/genética , Quimiocinas CXC , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exonucleases/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo
14.
Cell ; 129(2): 289-301, 2007 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-17412408

RESUMO

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.


Assuntos
Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular , Linhagem Celular , Dano ao DNA , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Mutação , Fase S , Strongylocentrotus purpuratus
15.
Science ; 316(5828): 1160-6, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17525332

RESUMO

Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Ciclo Celular/fisiologia , Linhagem Celular , Biologia Computacional , Sequência Consenso , Replicação do DNA/fisiologia , Humanos , Imunoprecipitação , Marcação por Isótopo , Camundongos , Células NIH 3T3 , Fosforilação , Proteoma/isolamento & purificação , Proteoma/fisiologia , RNA Interferente Pequeno , Transdução de Sinais , Especificidade por Substrato
16.
Mol Cell ; 23(3): 331-41, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16885023

RESUMO

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Cromatina/metabolismo , Dano ao DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Instabilidade Genômica/fisiologia , Células HCT116 , Histonas/metabolismo , Humanos , Hidroxiureia/farmacologia , Mutação/genética , Mutação/fisiologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ligação Proteica/efeitos da radiação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Fase S/fisiologia , Fase S/efeitos da radiação , Transfecção , Raios Ultravioleta
17.
Cell ; 122(4): 579-91, 2005 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-16122425

RESUMO

The BCL-2 family of apoptotic proteins encompasses key regulators proximal to irreversible cell damage. The BH3-only members of this family act as sentinels, interconnecting specific death signals to the core apoptotic pathway. Our previous data demonstrated a role for BH3-only BID in maintaining myeloid homeostasis and suppressing leukemogenesis. In the absence of Bid, mice accumulate chromosomal aberrations and develop a fatal myeloproliferative disorder resembling chronic myelomonocytic leukemia. Here, we describe a role for BID in preserving genomic integrity that places BID at an early point in the path to determine the fate of a cell. We show that BID plays an unexpected role in the intra-S phase checkpoint downstream of DNA damage distinct from its proapoptotic function. We further demonstrate that this role is mediated through BID phosphorylation by the DNA-damage kinase ATM. These results establish a link between proapoptotic Bid and the DNA-damage response.


Assuntos
Apoptose/genética , Proteínas de Transporte/metabolismo , Dano ao DNA/genética , Células Progenitoras Mieloides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Genes cdc/efeitos dos fármacos , Genes cdc/fisiologia , Instabilidade Genômica/genética , Leucemia Mielomonocítica Crônica/genética , Leucemia Mielomonocítica Crônica/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutagênicos/farmacologia , Células NIH 3T3 , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Fase S/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
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