Purificação e caracterização da VDAC de mitocôndrias corticais aviares: identificação de modificações pós-traducionais nas porinas neuronais murinas e aviares / Purification and characterization of avian cortical mitochondrial VDAC: identification of post-translational modifications of rat and avian neuronal porins

A VDAC é uma porina presente na MME cuja função é crucial no metabolismo energético, sobrevivência e morte celular. A caracterização da VDAC torna-se importante para a compreensão das inter-relações da mitocôndria com os diferentes componentes citosólicos, tais como a HK. A ligação HK-VDAC favorece a utilização do ATP intramitocondrial em células neuronais, a HK cerebral pode interagir de formas diferentes com a VDAC, o que resulta em diferentes sítios de ligação (sítios A e B). Os variados papéis metabólicos das isoformas da VDAC podem ser explicados pela presença de alterações pós-traducionais. No presente trabalho purificamos a VDAC1 mitocondrial neuronal proveniente de cérebro aviar. Paralelamente, comprovamos que a presença de múltiplas formas das VDACs 1 e 2 em cérebros murino e aviar, seja devida à presença de modificações pós-traducionais, nomeadamente a fosforilação. A proteína isolada apresentou peso molecular de 30KDa. Quando submetida à eletroforese e posteriormente à coloração para a identificação de fosfoproteínas, a mesma mostrou-se desfosforilada. O conhecimento da presença, ou ausência de fosforilação das VDACs, reside na importância de estabelecer-se as bases moleculares ligadas à existência de sítios A e B nas mitocôndrias neuronais.

VDAC (voltage-dependent anion channel) is a pore forming protein from outer mitochondrial membrane. It has key functions on energetic metabolism, and cell death and survival. VDAC characterization is important for understanding mitochondrial interactions with cytosolic proteins, such as hexokinase (HK). HK-VDAC interaction supports preferential access to intramitochondrial ATP in neural cells. Brain HK interacts in different ways with VDAC. It results in two HK binding sites (A and B). VDAC isoforms differential metabolic roles may be explained by the presence of post-translational modifications. In this study we purified avian neuronal mitochondrial VDAC1. At same time we showed that VDACs 1 and 2 pI heterogeneity in rat and avian brains is due to phosphorylation. Purified VDAC had a molecular weight of 30 KDa. The purified VDAC submitted to phosphorylated protein staining on gel, was dephosphorylated. The knowledge of presence or absence of VDAC phosphorylation is important for understanding the molecular nature basis of A and B HK binding sites in brain mitochondria.

Human Bop is a novel BH3-only member of the Bcl-2 protein family

One group of Bcl-2 protein family, which shares only the BH3 domain (BH3-only), is critically involved in the regulation of programmed cell death. Herein we demonstrated a novel human BH3-only protein (designated as Bop) which could induce apoptosis in a BH3 domain-dependent manner. Further analysis indicated that Bop mainly localized to mitochondria and used its BH3 domain to contact the loop regions of voltage dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane. In addition, purified Bop protein induced the loss of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c. Furthermore, Bop used its BH3 domain to contact pro-survival Bcl-2 family members (Bcl-2, Bcl-X(L), Mcl-1, A1 and Bcl-w), which could inhibit Bop-induced apoptosis. Bop would be constrained by pro-survival Bcl-2 proteins in resting cells, because Bop became released from phosphorylated Bcl-2 induced by microtubule-interfering agent like vincristine (VCR). Indeed, knockdown experiments indicated that Bop was partially required for VCR induced cell death. Finally, Bop might need to function through Bak and Bax, likely by releasing Bak from Bcl-X(L) sequestration. In conclusion, Bop may be a novel BH3-only factor that can engage with the regulatory network of Bcl-2 family members to process intrinsic apoptotic signaling.