RESUMO
DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA1/deficiência , Proteínas de Ciclo Celular/genética , Reparo do DNA/genética , Reparo de DNA por Recombinação/genética , ATPases Associadas a Diversas Atividades Celulares/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Humanos , Proteínas Mad2/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/efeitos dos fármacos , Proteínas de Ligação a Telômeros/genéticaRESUMO
Shelterin, a six-protein complex, plays a fundamental role in protecting both the length and the stability of telomeres. Repressor activator protein 1 (RAP1) and telomeric repeat-binding factor 2 (TRF2) are two subunits in shelterin that interact with each other. Small-molecule inhibitors that block the RAP1/TRF2 protein-protein interaction can disrupt the structure of shelterin and may be employed as pharmacological tools to investigate the biology of shelterin. On the basis of the cocrystal structure of RAP1/TRF2 complex, we have developed first-in-class triazole-stapled peptides that block the protein-protein interaction between RAP1 and TRF2. Our most potent stapled peptide binds to RAP1 protein with a Ki value of 7 nM and is >100 times more potent than the corresponding wild-type TRF2 peptide. On the basis of our high-affinity peptides, we have developed and optimized a competitive, fluorescence polarization (FP) assay for accurate and rapid determination of the binding affinities of our designed compounds and this assay may also assist in the discovery of non-peptide, small-molecule inhibitors capable of blocking the RAP1/TRF2 protein-protein interaction.
Assuntos
Proteínas de Ligação a Telômeros/efeitos dos fármacos , Proteína 2 de Ligação a Repetições Teloméricas/efeitos dos fármacos , Cristalografia por Raios X , Corantes Fluorescentes , Humanos , Modelos Moleculares , Mutação/genética , Peptídeos/síntese química , Peptídeos/farmacologia , Conformação Proteica , Complexo Shelterina , Bibliotecas de Moléculas Pequenas , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
It is well known that sulphonylureas (SUs), commonly used in the treatment of type 2 diabetes mellitus, stimulate insulin secretion by closing ATP-sensitive K(+) (K(ATP) ) channels in pancreatic ß-cells by binding to the SU receptor SUR1. SUs are now known also to activate cAMP sensor Epac2 (cAMP-GEFII) to Rap1 signalling, which promotes insulin granule exocytosis. For SUs to exert their full effects in insulin secretion, they are required to activate Epac2 as well as to inhibit the ß-cell K(ATP) channels. As Epac2 is also necessary for potentiation of glucose-induced insulin secretion by cAMP-increasing agents, such as incretin, Epac2 is a target of both cAMP and SUs. The distinct effects of various SUs appear to be because of their different actions on Epac2/Rap1 signalling as well as K(ATP) channels. Differently from other SUs, gliclazide is unique in that it is specific for ß-cell K(ATP) channel and does not activate Epac2.
Assuntos
Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipoglicemiantes/uso terapêutico , Células Secretoras de Insulina/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Receptores de Droga/efeitos dos fármacos , Compostos de Sulfonilureia/uso terapêutico , Transportadores de Cassetes de Ligação de ATP/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Canais KATP/efeitos dos fármacos , Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Droga/metabolismo , Complexo Shelterina , Transdução de Sinais/efeitos dos fármacos , Receptores de Sulfonilureias , Proteínas de Ligação a Telômeros/efeitos dos fármacos , Proteínas de Ligação a Telômeros/metabolismoAssuntos
Anticolesterolemiantes/uso terapêutico , Doença das Coronárias/prevenção & controle , Pravastatina/uso terapêutico , Telômero/efeitos dos fármacos , Doença das Coronárias/etiologia , Humanos , Masculino , Prognóstico , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Telômero/fisiologia , Proteínas de Ligação a Telômeros/sangue , Proteínas de Ligação a Telômeros/efeitos dos fármacosRESUMO
The effects on S.cerevisiae telomere binding protein Rap1p, telomerase and telomeric DNA by the lead (Pb), the selenium (Se) and Pb + Se were tested respectively in this study. Compared with the control S.cerevisiae after 100 gene rations, the mean telomere length shortened, Rap1p concentration was significantly lower and the secondary structure of Rap1p was disturbed, the telomerase activity was reduced in Pb treated cells. In Se treated cells, telomere length was significantly longer, and telomerase activity expressed higher. The concentration and secondary structure of Rap1p were similar to that of the control. Further more, the viability of Pb treated cells were significantly reduced while cells undergone other three treatments were similar and normal. These results suggest that Pb could damage Rap1p, reduce telomerase activity, resulting in the telomer length shortening and cell death. On the other hand, Se could protect and repair the damage in Rap1p and telomere caused by Pb to some extent.