RESUMO
Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
Assuntos
Proteínas de Ciclo Celular , Proteólise , Transdução de Sinais , Fatores de Transcrição , Ubiquitinação , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas que Contêm BromodomínioRESUMO
The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.
