RESUMO
Death receptor 5 (DR5) is a promising target for antitumor therapy due to its high expression on different tumor cells. Resistance of various tumor cells against TRAIL, a natural ligand for the death receptors, reduces its therapeutic potential and prompts the search for novel agonists at these receptors. Previous screening across the combinatorial peptide library yielded a peptide sequence KVVLTHR that specifically binds DR5. Incorporation of this sequence into TNFα resulted in binding DR5 with mutant protein TNFα-mut and appearance of cytotoxicity against lymphoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Linfócitos/efeitos dos fármacos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Apoptose/genética , Sítios de Ligação , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.
