Biological Function Analysis Of FKBP25 And Its Underlying Molecular Mechanism During Cerebral Ischemia

Background:Stroke is one of the serious diseases that hazardous human health,which has high morbidity,disability and mortality rate.Ischemia stroke constitutes 60%to 70%of the total stroke.However,we are still facing the effective drugs on clinical and effective drug-target deficiency for the treatments of ischemia stroke.Cerebral ischemic injury also influence on normal physiological function.The neurovascular unit is a complex organism which composed of a microvascular endothelium,neuron,and glial cell elements.It remains an impotant question to explore the bio-molecular events in neurovascular unit following brain ischemia.Peptidyl-prolyl cis-trans isomerase FKBP25 is a member of the FK506-binding proteins family which has peptidylprolyl cis/trans isomerase domain,Proline is the only imino acid of the 20 kinds of basic amino acids that provides the possibility for protein conformation change with cis/trans isomerization.Previous work showed peptidyl-prolyl cis-trans isomerase are widely expressed in many tissues,involved in key biochemical processes including protein folding,receptor signaling,protein trafficking and transcription.However,the biological fuction and pathophysiologic role of FKBP25 in brain diseases remain largelly unknown.Objective:The objective of present study is to explore the role of FKBP25 in neurovascular unit under cerebral ischemia.Firstly,we examine the biological activity and molecular mechanism of endothelial FKBP25 in the progress of ischemic injury.Secondly,to explore the pharmacological effects of FKBP25 isomerization activity on neurons.Finally,we design and examine a novel fluorescent probe to determine the activity of the peptidyl-prolyl cis-trans isomerase.Methods and Results:In the present study,western blot and immunofluorescence data revealed that FKBP25 can translocate from cytoplasm to nucleus.This phenomenon also confirmed by recording living cells upon ONOO-stimulation.The interacting proteins with FKBP25 were analyzed by using immunoprecipitation and mass spectra.We found that the 60S ribosomal protein L7 is the candidate protein interacting with FKBP25.Importantly,overexpression of FKBP25 reduced the endothelial cells damage following OGD(oxygen-glucose deprivation)injury,as indicated by the decreased PARP protein level and apoptosis rate.The breakdown of spectrin and calcineurin were increased after transient middle cerebral artery occlusion(tMCAO)on mice,whereas FKBP25 overexpression treatment attenuated the breakdown products of spectrin and calcineurin following tMCAO.We also observed the effect of FKBP25 on against leakage of blood-brain barrier(BBB)during brain ischemia.Furthermore,we shown that FKBP25 translocated into nucleus in which bind with 60S ribosomal protein L7,might related to its potential pharmacological effect.Whole-cell patch-clamp recording data demonstrated that the recombinant protein FKBP25 influenced the calcium current on pyramidal neurons from acute brain slices of mice.In the present study,a novel fluorescent probe(PPI-CT6)for determining peptidyl-prolyl cis-trans isomerase(PPIase)activity was designed and confirmed in endothelial cells.Conclusion:Our studies identify FKBP25 is an important and sensitive molecular which involved in cerebral ischemic injury.The abnormal nuclear import of FKBP25 and binding with 60S ribosomal protein L7 is likely associated with nitrosative stress response of endothelial cells following ischemia injury.Moreover,FKBP25 can regulate calcium current on pyramidal neurons which dependent on its enzyme activity.In the present study,we also developed the novel fluorescent probe which exhibit higher sensitivity and specificity to the peptidyl-prolyl cis-trans isomerase.