RESUMO
BACKGROUND: We have identified endogenous p65 to be an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. We have investigated molecular properties involved in the stability of dimeric form, and their regulation in the transition between monomeric and dimeric forms of hnRNPA/B-like protein 2. We also investigated a cellular property conserved between squid hnRNPA/B-like protein 2 and human hnRNPA1 protein in a neuronal context. METHODS AND RESULTS: Here we show biochemical properties of a recombinant hnRNPA/B-like protein 2 (rP2) in vitro experiments, as one of p65 subunit. We found that interaction between rP2 and RNA molecules interfered with the dynamics of rP2 dimers formation, involved in disulfide bonds and/or postranslational alterations in distinct stage of SDS-stable dimers formation. In addition, we have performed immunofluorescence in SH-SY5Y cells and observed that the pEGFP-P2 fusion protein was expressed in the nucleus, similar to what is observed for human hnRNPA1 protein. CONCLUSION: Our results reinforce the idea that p65 is an SDS-stable dimer. Thus, a deeper understanding between monomeric and dimeric transition dynamic is critical into evolution of several neurodegenerative disease.
Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Animais , Decapodiformes/genética , Decapodiformes/metabolismo , Humanos , Pressão Osmótica , Proteínas Recombinantes/genéticaRESUMO
In previous works, we identified a RNA-binding protein in presynaptic terminal of squid neurons, which is likely involved in local mRNA processing. Evidences indicate this strongly basic protein, called p65, is an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. The function of p65 in presynaptic regions is not well understood. In this work, we showed p65 and its subunit p37 are concentrated in RNA-enriched regions in synaptosomes. We performed in vitro binding studies with a recombinant protein and showed its propensity to selectively bind actin mRNA at the squid presynaptic terminal. Biochemical analysis using lysed synaptosomes suggested RNA integrity may affect p65 and p37 functions. Mass spectrometry analysis of oligo(dT) pull down indicated squid hnRNPA1, hnRNPA1-like 2, hnRNPA3 and ELAV-like proteins as candidates to interact with p65 and p37 forming a ribonucleoprotein complex, suggesting a role of squid hnRNPA/B-like proteins in site-specific RNA processing.