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1.
Mol Cell ; 29(4): 451-64, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18313383

RESUMO

The tumor suppressor CYLD antagonizes NF-kappaB and JNK signaling by disassembly of Lys63-linked ubiquitin chains synthesized in response to cytokine stimulation. Here we describe the crystal structure of the CYLD USP domain, revealing a distinctive architecture that provides molecular insights into its specificity toward Lys63-linked polyubiquitin. We identify regions of the USP domain responsible for this specificity and demonstrate endodeubiquitinase activity toward such chains. Pathogenic truncations of the CYLD C terminus, associated with the hypertrophic skin tumor cylindromatosis, disrupt the USP domain, accounting for loss of CYLD catalytic activity. A small zinc-binding B box domain, similar in structure to other crossbrace Zn-binding folds--including the RING domain found in E3 ubiquitin ligases--is inserted within the globular core of the USP domain. Biochemical and functional characterization of the B box suggests a role as a protein-interaction module that contributes to determining the subcellular localization of CYLD.


Assuntos
Lisina/metabolismo , Poliubiquitina/metabolismo , Estrutura Terciária de Proteína , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Enzima Desubiquitinante CYLD , Genes Supressores de Tumor , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Neoplasias/genética , Poliubiquitina/química , Poliubiquitina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Especificidade por Substrato , Proteínas Supressoras de Tumor/classificação , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina , Zinco/metabolismo
3.
EMBO J ; 27(9): 1368-77, 2008 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-18388863

RESUMO

Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.


Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , RNA Interferente Pequeno/genética , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Dano ao DNA , Reparo do DNA , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Proteínas Supressoras de Tumor
4.
EMBO Rep ; 10(9): 990-6, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19609323

RESUMO

The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy-terminal beta-propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein-protein interaction and explains the effects of both cancer-associated truncating mutants in PALB2 and missense mutations in the amino-terminal region of BRCA2.


Assuntos
Proteína BRCA2/química , Proteína BRCA2/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Proteína BRCA2/genética , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Proteína do Grupo de Complementação N da Anemia de Fanconi , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteínas Supressoras de Tumor/genética
5.
DNA Repair (Amst) ; 7(12): 2010-9, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18832051

RESUMO

Synthetic lethality is an attractive strategy for the design of novel therapies for cancer. Using this approach we have previously demonstrated that inhibition of the DNA repair protein, PARP1, is synthetically lethal with deficiency of either of the breast cancer susceptibility proteins, BRCA1 and BRCA2. This observation is most likely explained by the inability of BRCA deficient cells to repair DNA damage by homologous recombination (HR) and has led to the clinical trials of potent PARP inhibitors for the treatment of BRCA mutation-associated cancer. To identify further determinants of PARP inhibitor response, we took a high-throughput genetic approach. We tested each of the genes recognised as having a role in DNA repair using short-interfering RNA (siRNA) and assessed the sensitivity of siRNA transfected cells to a potent PARP inhibitor, KU0058948. The validity of this approach was confirmed by the identification of known genetic determinants of PARP inhibitor sensitivity, including genes involved in HR. Novel determinants of PARP inhibitor response were also identified, including the transcription coupled DNA repair (TCR) proteins DDB1 and XAB2. These results suggest that DNA repair pathways other than HR may determine sensitivity to PARP inhibitors and highlight the likelihood that ostensibly distinct DNA repair pathways cooperate to maintain genomic stability and cellular viability. Furthermore, the identification of these novel determinants may eventually guide the optimal use of PARP inhibitors in the clinic.


Assuntos
Enzimas Reparadoras do DNA/antagonistas & inibidores , Reparo do DNA , Fluorbenzenos/farmacologia , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , RNA Interferente Pequeno/genética , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco
6.
Oncotarget ; 5(23): 12126-40, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25565632

RESUMO

CYLD, an ubiquitin hydrolase, has an expanding repertoire of regulatory roles in cell signalling and is dysregulated in a number of cancers. To dissect CYLD function we used a proteomics approach to identify CYLD interacting proteins and identified MIB2, an ubiquitin ligase enzyme involved in Notch signalling, as a protein which interacts with CYLD. Coexpression of CYLD and MIB2 resulted in stabilisation of MIB2 protein levels and was associated with reduced levels of JAG2, a ligand implicated in Notch signalling. Conversely, gene silencing of CYLD using siRNA, resulted in increased JAG2 expression and upregulation of Notch signalling. We investigated Notch pathway activity in skin tumours from patients with germline mutations in CYLD and found that JAG2 protein levels and Notch target genes were upregulated. In particular, RUNX1 was overexpressed in CYLD defective tumour cells. Finally, primary cell cultures of CYLD defective tumours demonstrated reduced viability when exposed to γ-secretase inhibitors that pharmacologically target Notch signalling. Taken together these data indicate an oncogenic dependency on Notch signalling and suggest potential novel therapeutic approaches for patients with CYLD defective tumours.


Assuntos
Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Enzima Desubiquitinante CYLD , Células HEK293 , Humanos , Imuno-Histoquímica , Imunoprecipitação , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/metabolismo , Análise Serial de Tecidos
7.
Cancer Res ; 66(16): 8109-15, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16912188

RESUMO

Deficiency in either of the breast cancer susceptibility proteins BRCA1 or BRCA2 induces profound cellular sensitivity to the inhibition of poly(ADP-ribose) polymerase (PARP) activity. We hypothesized that the critical role of BRCA1 and BRCA2 in the repair of double-strand breaks by homologous recombination (HR) was the underlying reason for this sensitivity. Here, we examine the effects of deficiency of several proteins involved in HR on sensitivity to PARP inhibition. We show that deficiency of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM, CHK1, CHK2, FANCD2, FANCA, or FANCC induces such sensitivity. This suggests that BRCA-deficient cells are, at least in part, sensitive to PARP inhibition because of HR deficiency. These results indicate that PARP inhibition might be a useful therapeutic strategy not only for the treatment of BRCA mutation-associated tumors but also for the treatment of a wider range of tumors bearing a variety of deficiencies in the HR pathway or displaying properties of 'BRCAness.'


Assuntos
Dano ao DNA , Reparo do DNA , Inibidores de Poli(ADP-Ribose) Polimerases , Recombinação Genética , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Fluorbenzenos/farmacologia , Camundongos , Camundongos Knockout , Ftalazinas/farmacologia , Células-Tronco/citologia
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