Mechanism Of Antimicrobial Peptide CGA-N12 On Mitochondrial Permeability Transition Pore Of Candida Tropicalis

Antimicrobial peptide CGA-N12 is a peptide derived from the N-terminus of human chromogranin A?CGA 65-76?,which has specific anticandidal activity and no toxicity to mammalian cells.Our previous research demonstrated that CGA-N12 induces membrane potential dissipation.However,the mechanism of CGA-N12 reducing mitochondrial membrane potential remains unclear.Mitochondrial permeability transition pores?mPTP?is the regulation center of mitochondrial activity.The effect of CGA-N12 on mPTP was studied to reveal the mechanism of CGA-N12 reducing mitochondrial membrane potential.The results of transmission electron microscopy and spectroscopy showed that CGA-N12 changed the mitochondrial ultrastructure,and indueced the matrix swollen,indicating that mitochondrial membrane permeability increased;the substances below 1500Da passed through mitochondria membrance freely;CGA-N12 promoted the release of cytochrome c?Cyt c?and Ca²⁺,which demonstrated that mPTP opened irreversibly.The followed proton gradient disappear and the membrane potential dissipation caused the mitochondrial ATPase hydrolysis activity to increase,which aggravated mPTP open.Therefore,CGA-N12 reduced mitochondrial membrane potential of C.tropicalis by opening mPTP.The action sites of CGA-N12 on mPTP protein complex were studied by using mPTP-opening inhibitors-cyclosporin A?CsA?,bongkrekic acid?BA?,monobromobimane?MBM⁺?,and dithiothreitol?DTT?.The results showed that BA and MBM⁺completely and DTT partially prevented mPTP opening caused by CGA-N12.Therefore,CGA-N12 opens mPTP by attacking the matrix?m?state of adenine nucleotide transposase?ANT?as well as maintaining the crosslink of disulfide linkage of mPTP protein complex on the intermembrane surface on the inner mitochondrial membrane and oxidizing free-SH groups of mPTP complex proteins in mitochondria.The oxidation may be related to the large accumulation of ROS in C.tropicalis cell induced by CGA-N12.To clarify the specificity of CGA-N12 on Candida cells,the composition of mPTP complex main proteins between C.tropicalis cells and mammal cells were compared.Genomic DNA of normal human hepatocyte cell line L02 and C.tropicalis were used as templates to perform PCR amplification of the conserved sequences of each protein gene.It was found that the C.tropicalis genomic DNA was amplified with the conserved sequence primers of mammalian cells m PTP complex protein gene,CyPD,VDAC I?III subtypes and ANT I?IV subtype genes could be amplified,but BAX and Bcl-2 genes were not amplified;L02 cells genomic DNA was amplified with the conserved gene primers of fungal mPTP complex protein genes,and CyPD and VDAC genes were amplified.The results demonstrated that C.tropicalis genomic DNA had no BAX and Bcl-2 protein genes,and contained CyPD,VDAC and ANT protein genes as well as the amplification sites of human CyPD,VDAC I?III subtype,ANT I?IV subtype conserved gene sequence primers;L02 cells genomic DNA contained the amplification sites of the fungal CyPD and VDAC conserved gene sequence primers,without fungal ANT primer.The results initially showed that the composition of candida and mammalian mPTP are different,and the specific differences between them in proteins need further study.In summary,CGA-N12 opens mPTP via destroying the conformation of the ANT matrix,maintaining the disulfide bond and oxidizing free-SH groups on mPTP,resulting in mitochondrial membrane potential dissipation.Compared with mammalian mPTP,C.tropicalis mPTP has no the conserves genes of apoptosis regulators BAX and Bcl-2,and contains CyPD,VDAC and ANT protein genes.