RESUMO
Three JNK isoforms, JNK1, JNK2, and JNK3 have been reported and unique biological function has been ascribed to each. It is unknown if selective inhibition of these isoforms would confer therapeutic or safety benefit. To probe JNK isoform function we designed JNK2/3 inhibitors that have >30-fold selectivity over JNK1. Utilizing site-directed mutagenesis and x-ray crystallography we identified L144 in JNK3 as a key residue for selectivity. To test whether JNK2/3 selective inhibitors protect human dopaminergic neurons against neurotoxin-induced mitochondrial dysfunction, we monitored reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP). The results showed that JNK2/3 selective inhibitors protected against 6-hydroxydopamine-induced ROS generation and MMP depolarization. These results suggest that it was possible to develop JNK2/3 selective inhibitors and that residues in hydrophobic pocket I were responsible for selectivity. Moreover, the findings also suggest that inhibition of JNK2/3 likely contributed to protecting mitochondrial function and prevented ultimate cell death.
Assuntos
Proteína Quinase 10 Ativada por Mitógeno/química , Proteína Quinase 9 Ativada por Mitógeno/química , Inibidores de Proteínas Quinases/química , Pirazóis/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/genética , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxidopamina/farmacologia , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirazóis/metabolismo , Pirazóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
In response to NMDA receptor stimulation, CaMKIIα moves rapidly from a diffuse distribution within the shafts of neuronal dendrites to a clustered postsynaptic distribution. However, less is known about CaMKIIα localization and trafficking within neuronal somata. Here we use a novel recombinant probe capable of labeling endogenous CaMKIIα in living rat neurons to examine its localization and trafficking within the somata of cortical neurons. This probe, which was generated using an mRNA display selection, binds to endogenous CaMKIIα at high affinity and specificity following expression in rat cortical neurons in culture. In â¼45% of quiescent cortical neurons, labeled clusters of CaMKIIα 1-4 µm in diameter were present. Upon exposure to glutamate and glycine, CaMKIIα clusters disappeared in a Ca(2+)-dependent manner within seconds. Moreover, minutes after the removal of glutamate and glycine, the clusters returned to their original configuration. The clusters, which also appear in cortical neurons in sections taken from mouse brains, contain actin and disperse upon exposure to cytochalasin D, an actin depolymerizer. In conclusion, within the soma, CaMKII localizes and traffics in a manner that is distinct from its localization and trafficking within the dendrites.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Actinas/metabolismo , Animais , Células COS , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Citocalasina D/farmacologia , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Camundongos , Transporte Proteico/efeitos dos fármacos , RatosRESUMO
The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.
