Rakhmetov A. Identification of the specific interaction between peroxiredoxins and CKBB proteins under stress conditions

To investigate functional reactivity of the recombinant CKBB protein heat and hydrogen peroxide stresses were applied. The results of specific enzymatic assay have indicated on high susceptibility of the recombinant CKBB to stressors. Reduction of specific activity of CKBB under 1 mM hydrogen peroxide was registered by 74% and heat 42°C by 86%. To support our assay data heat and hydrogen peroxide stresses we applied to A549 and HeLa cells expressing CKBB. Under the stresses reduced CKBB immunoblot signals were registered. This results suggest low resistibility of CKBB to heat and hydrogen peroxide stresses. The results of co-immunoprecipitation assay (IP) have shown that Prx I has weak binding with of CKBB whereas Prx II demonstrated strong interaction with CKBB in A549 and HeLa cells. Exposure to heat stress but not to hydrogen peroxide resulted in increased interaction signal between Prx II and CKBB protein. To understand whether truncation of C-terminal region of Prx II would affect the interaction of Prx II and CKBB specific mutants of Prx II were introduced into the HeLa and A549 cells. IP results have indicated on relatively high binding affinity between Prx II truncated mutants and CKBB. To investigate chaperoning properties of Prx I and Prx II we used CKBB phosphorylation assay. We found that chaperoning capacity of Prx I under elevated temperature was concentration-dependent. At 3 µM Prx I preserved 95% of CKBB specific activity what showed better protection results than Prx II. In contrast, Prx II was considerably more effective than Prx I in protection of CKBB specific activity under hydrogen peroxide stress.