Study Of The In Vitro Enzyme Activity Of PINK1,a Parkinson’s Disease-associated Protein

Mitochondrial dysfunction is one of the causes of Parkinson’s Disease(PD).The mitochondrial kinase PINK1,which is located on the outer mitochondrial membrane,participates in mitophagy,and its mutations can cause familial,autosomal recessive Parkinson’s disease.PINK1 is encoded by PD-related pathogenic gene PARK6,and its topology includes: N-terminal mitochondrial localization domain,single transmembrane domain,kinase domain,and C-terminal domain.From the crystal structural information of PINK1 from Pediculus humanus corporis and Tribolium castaneum,it can be found that there is a close interaction between the C-terminal domains of PINK1,as well as between C-terminal domain and the C-lobe of the kinase domain.However,the function of the C-terminal domain of PINK1,its role in the regulation of kinase activity,and how it regulates kinase activity are still unknown.This study developed a novel method for detecting the kinase activity of PINK1.Through this method,the kinase activity of PINK1 can be detected quantitatively in vitro with high sensitivity by labeling FITC fluorescent probe on Ubiquitin(Ub),the substrate of PINK1.Using this method,we measured the enzyme kinetic parameters of TcPINK1 and the half-inhibition constant of inhibitor A for TcPINK1.In order to explore the regulatory mechanism of PINK1 activity,we analyzed the structural information of PINK1.According to the structural analysis,we speculate that PINK1 will form a dimer through the interaction between C-terminal domains,which may have an important regulatory function for the kinase activity.To verify this conjecture,we designed and constructed several mutants to disrupt the interaction between the C-terminal domains of TcPINK1.By measuring and comparing the aggregation state and kinase activity of wild-type TcPINK1 and the mutants,we found that the interaction of the C-terminus of TcPINK1 has a regulatory effect on stabilizing the dimer structure of PINK1 and inhibiting the binding of PINK1 to substrate.The above results are of great theoretical significance for laying a foundation for elucidating the biological function and kinase regulatory mechanism of PINK1.