Mitochondrial 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNP) interacts with mPTP modulators and functional complexes (I-V) coupled with release of apoptotic factors

We previously reported that 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) is present in rat brain and liver mitochondria, in the outer membrane and mitoplasts. Substrates of CNP, 2′,3′-cAMP and 2′,3′-cNADP, were found to accelerate opening of mitochondrial permeability transition pore (mPTP). In purified non-synaptic mitochondria, CNP was observed to co-immunoprecipitate with main modulators of mPTP, i.e. VDAC, ANT, and cyclophilin D, as well as with tubulin and COX IV. Using Blue Native Electrophoresis, with following Western blot, CNP was revealed to associate with functional inner membrane mitochondrial complexes I-V. In Ca2+ -overloaded mitochondria, association of CNP with complexes I-V was decreased. Cyclosporine A increased the association of CNP with complexes I and III, supporting the idea of the involvement of these complexes in mPTP function. 2′,3′-cAMP enhanced CNP dissociation from complexes I, III, IV and V in Ca2+-overloaded mitochondria (i.e. when pore is opened). Association of CNP with complexes I, III, IV, and V was shown in mitochondria isolated from brain, liver and heart. Stimulation of the opening of the non-selective pore in mitochondria correlated with CNP release from mitochondria in parallel with release of cytochrome c, AIF and Endo G. In Ca2+-overloaded mitochondria, 2′,3′-cAMP further accelerated the release of AIF, Endo G and CNP, but did not alter cytochrome c release. These results provide strong evidence that CNP, one of the possible regulators of mPTP complex, might be involved in the control of respiration and energy production in mitochondria. This reveals a new function of CNP outside the myelin structure.