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

Yulia Baburuna,Linda Sotnikova,Olga Krestinina

doi:10.3390/ijms221910613

Abstract

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.

Highlights

Phosphorylation of proteins is one of the most common and important post-translational modifications involved in the regulation of many processes in various cellular compartments, including mitochondria [1]
We showed that in Ca2+ over-loaded rat brain mitochondrial (RBM) in the presence of Mg-ATP several membrane-bound mitochondrial proteins were able to be phosphorylated by protein kinase
The proteins with molecular weights of 17 and 21 kDa were identified as myelin basic protein [8], 46–48 kDa protein as 2,3 -cyclyc nucleotide -3 -phosphodiesterase (CNPase) [7], and 3.5 kDa phosphoprotein as subunit c of FoF1-ATPase [9]

Summary

Introduction

Phosphorylation of proteins is one of the most common and important post-translational modifications involved in the regulation of many processes in various cellular compartments, including mitochondria [1]. Phosphorylation/dephosphorylation of proteins is an important signal transduction regulatory system that controls many aspects of cellular functions that can affect the properties of enzymatic activity, structure, subcellular localization, and stability of some proteins [3]. The level of protein phosphorylation in RBM was found to be altered in the presence of threshold Ca2+ concentration, leading to opening of mPTP. The results showed a clear correlation between mPTP opening and changes in the level of protein phosphorylation in RBM [5]. We found a clear correlation between mPTP function and changes in the level of phosphorylation of 60–62, 46–48, 17, 3.5 kDa proteins in RBM and suggested that protein phosphorylation is an important step in the regulation of mPTP function [5]

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