Assuntos
Células da Medula Óssea/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcriptoma , Animais , Antineoplásicos/farmacologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Técnicas de Cocultura , Citarabina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Neurônios/metabolismo , Neurônios/patologia , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recidiva , Transdução de Sinais , Transplante Heterólogo , Microambiente Tumoral/genéticaRESUMO
We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa's BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel ß-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.
Assuntos
Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Simulação de Dinâmica Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Peptídeos , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
PURPOSE: The purpose of this study was to develop methods to allow evaluation of the binding characteristics for a series of α-1 antagonists to biologically-derived melanin. METHODS: Fresh bovine globes were used to obtain iridal and choroid/retinal pigment epithelial (CRPE) derived melanin. Binding characteristics of chloroquine, tamsulosin and doxazosin were then evaluated in vitro using tandem mass spectroscopy. RESULTS: Tandem mass spectrometry-based assays were developed for three α-1 antagonists that provided linear assay ranges which spanned (minimally) 0.01-10 µg/mL, while exhibiting excellent inter-assay precision and accuracy. When applied to the evaluation of binding characteristics for iridal melanin, mean chloroquine and tamsulosin fractions were found to be 41.9 ± 14.2 pmoles mg(-1) and 25.34 ± 6.186 pmoles mg(-1), respectively. Mean iridal doxazosin binding was found to be 6.36 ± 2.19 pmoles mg(-1). Interestingly, mean levels of tamsulosin, but not doxazosin found bound to choroid/CRPE derived melanin approached that of chloroquine (27.91 µg/mL, 25.68 µg/mL and 5.94 µg/mL for chloroquine, tamsulosin and doxazosin, respectively). One way ANOVA for binding affinity for chloroquine, tamsulosin and doxazosin was statistically significant for both iridal and CRPE-derived melanin (p = 0.0012 and 0.0023), respectively. A Bonferroni post-hoc analysis demonstrated a statistically significant difference in the amount of binding between tamsulosin, doxazosin and chloroquine to iridal but not CRPE derived melanin (p < 0.05). CONCLUSIONS: Tamsulosin appears to demonstrate melanin binding affinity which approaches chloroquine and exceeds doxazosin for both iridal and CRPE-derived bovine melanin.
