Study On Changes Of Mitochondrial Function And The Mechanism During PMA-induced K562 Megakaryocytic Differentiation

Proteins usually exert their function by forming protein complexs. The cell nucleus is the center of gene expression and metabolic regulation that contains a number of important protein complexes, such as the spliceosome, the nuclear pore complex, etc. The mitochondria play critical roles in the maintenance of cellular energy suppliement, and perform oxidative phosphorylation, the energy-generating processes coupling the oxidation of substrates to the synthesis of nearly all cellular ATP. In addition, mitochondria are involved in the synthesis of reactive oxygen species (ROS), apoptosis and metabolism. However, there are rare report about nuclear protein complexes especially the interrelation between each complex, and the dynamics of mitochondrial respiratory chain complexs during cell differentiation.In this study, we established and optimized the blue native electrophoresis (BN-PAGE) followed by mass spectrum technique policy to separate and identify the protein complexes in human K562 erythroleukemia cell line, 12 protein complexes were separated and identified. Detailed analysis showed that the complex 1 containes 40 proteins, including 8 sub-components of CCT complex, some sub-components of RNA spliceosome, some proteins related to cell growth and apoptosis and etc. From the bioinformatic analysis, there were 35 of 40 proteins (87.5%) have known interactions. As expected, some known CCT binding partners were identi?ed, such as TUBA4A and TUBA8. BN-PAGE followed by native immunoblotting analysis were performed to validate the interactions in complexs 1 and discovered 5 identified proteins in the same complex with CCT complex. The essential role of CCT complex is to maintain cellular homoeostasis by assisting the folding of many newly synthesized proteins in eukaryotic cells. Our results indicate that the CCT complex may assist some protein complexes aggregation and involve in RNA processing/RNA splicing in nucleus, which provide new avenues of inquiry inro CCT complex functions.Further, BN-PAGE combined in-gel catalytic activity assays were performed to investigate the dynamics of mitochondrial respiratory chain complex and the relationship with mitochondrial function during PMA-induced megakaryocytic differentiation in K562 cells. The results showed that the mitochondrial function damaged remarkably during megakaryocytic differentiation. Second, in the presence of cyclosporin A which stabilizes the mitochondrial membrane potential, the PMA-induced K562 megakaryocytic differentiation was improved. Third, during K562 differentiation, the activity of complexâ…£of mitochondrial respiratory chain reduced primely then increased. Fourth, treatment of the inhibitor of complexâ…£could blocks K562 differentiation. Cyclosporin A did not prevent the drop of the activity of complexâ…£. Fifth, during K562 differentiation, the expression of COX3, the core subunit of complexâ…£, and mitochondrial translocase proteins Tim9 and Tim10 were decreased. These results indicated that mitochondrial function was changed during PMA-induced K562 megakaryocytic differentiation, the fall of the activity of complexâ…£induced the drop in mitochondrial membrane potential, and eventually resulted in the injury of mitochondrial function. Consequently, the stabilization of mitochondrial function is important cell differentiation.