Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60–62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca2+ efflux from mitochondria, preventing the opening of mPTP. It was found that phosphorylated truncated CALP3 with a molecular weight of 60–62 contains p-Tyr, which indicates the possible involvement of protein tyrosine phosphatase in this process.

Single-walled and multiwalled carbon nanotubes induce oxidative stress in isolated rat brain mitochondria

Single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs) are broadly applicable across a variety of industrial fields. Despite their usefulness in many different applications, oxidative stress-induced toxicity of SWCNTs and MWCNTs has not been widely investigated. The present study examined the effects of SWCNTs and MWCNTs on rat brain mitochondria using the 3,4 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and indices of reactive oxygen species (ROS), based on measurements of malondialdehyde (MDA), glutathione (GSH), and mitochondrial membrane potential. Based on the MTT assay, exposure to SWCNTs and MWCNTs decreased mitochondrial survival and viability in a dose-dependent manner. Findings also indicated that MWCNTs and SWCNTs could damage mitochondrial membranes and induce the formation of ROS, as indicated by increased levels of MDA and decreased GSH content. The results of this study suggest that SWCNTs and MWCNTs likely damage brain tissue mitochondria by increasing oxidative stress and possibly activating the apoptosis pathway as well as other pathways of cytotoxicity.

Possible Involvement of 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase in the Protein Phosphorylation-Mediated Regulation of the Permeability Transition Pore

Calcium as a secondary messenger regulates the phosphorylation of several membrane-bound proteins in brain and liver mitochondria. Regulation of the activity of different protein kinases and phosphatases by Ca2+ occurs through its binding with calmodulin. The protein phosphorylation is strongly dependent on the Ca2+-induced mitochondrial permeability transition pore (mPTP) opening. 2′,3′-Cyclic nucleotide-3′-phosphodiesterase (CNPase) was phosphorylated by protein kinases A and C. CNPase and melatonin (MEL) might interact with calmodulin. The effects of the calmodulin antagonist calmidazolium and the inhibitor of protein kinase A H89 on mPTP opening in rat brain mitochondria of male Wistar rats were investigated. In addition, the role of CNPase, serine/threonine kinases, and MEL in the mPTP opening was examined. The anti-CNPase antibody added to rat brain mitochondria (RBM) reduced the content of CNPase in mitochondria. The threshold [Ca2+] decreased, and mitochondrial swelling was accelerated in the presence of the anti-CNPase antibody. H89 enhanced the effect of anti-CNPase antibody and accelerated the swelling of mitochondria, while CmZ abolished the effect of anti-CNPase antibody under mPTP opening. The levels of phospho-Akt and phospho-GSK3β increased, while the MEL content did not change. It can be assumed that CNPase may be involved in the regulation of these kinases, which in turn plays an important role in mPTP functioning.