Molecular Mechanisms And Regulatory Mechanisms In PINK1-Parkin Mediated Mitophagy

Mitophagy,a mechanism for the selective removal of damaged mitochondria through autophagy,plays a key role in the pathogenesis of neurodegenerative diseases.PINK1-Parkin-mediated mitophagy is the most typical mitophagy pathway.PINK1 is first activated by aggregation on the mitochondrial outer membrane,and then phosphorylates ubiquitin,which recruits Parkin to the mitochondria.Under the action of PINK1,Parkin is activated and ubiquitinates mitochondrial outer membrane proteins to initiate mitophagy.At present,the catalytic mechanism of the kinase PINK1 during mitophagy,the regulatory mechanism of post-translational modification,and the dynamic structural mechanism of polyubiquitin chains are still insufficiently studied.Therefore,this thesis combined the advantages of NMR spectroscopy in protein structure analysis and protein dynamic structure research and the advantages of single-molecule fluorescence resonance energy transfer(sm FRET)in single-molecule level protein dynamic structure research to study the mitophagy pathway.The main contents are as follows:(1)In this thesis,it was found for the first time by mass spectrometry that PINK1 could phosphorylate Ub at S65 and T66 residues.The two solution conformations of p S65/p T66 Ub were further analyzed by nuclear magnetic resonance technology.Further NMR experiments showed that the additional p T66 phosphate group enhance the p H sensitivity of p Ub conformational switch through electrostatic mutual renting with neighboring K63.(2)A good model L50 A mutant for studying the conformational transition of ubiquitin was designed.It was found by NMR experiments that the number of two conformational states of Ub can be regulated by a single mutation,L50 A,which is more prone to transition to the contracted conformation.Combined with protein dynamics research methods,PINK1 was found to induce a conformational change of Ub when it catalyzes the substrate Ub.(3)In the study of the inhibitory mechanism of PINK1-Parkin-mediated mitophagy,PP2 A was found to mediate the dephosphorylation of p Parkin and p Ub by solution NMR.And identified by mass spectrometry that B55α is a regulatory subunit that accelerates the dephosphorylation of p Parkin and p Ub.The negative regulatory effect of PP2 A on PINK1-Parkin-mediated mitophagy was then demonstrated by fluorescence imaging.(4)Using the superiority of single-molecule fluorescence resonance energy transfer technology in capturing the dynamic changes of protein conformation at the single-molecule level,combined with fluorescence polarization technology,the dynamic structure of K48-linked polyubiquitin was studied.The regulation of the dynamic structure and function of K48-linked polyubiquitin by unstructured and structured substrate proteins was also explored.In summary,the kinase PINK1 was found in this thesis to simultaneously phosphorylate Ser65 and Thr66 of Ub,and it was found that multi-site phosphorylation enables the transition between the two conformational states of Ub to respond rapidly to p H.Then PINK1 was found to cause a conformational change of Ub when catalyzing the substrate Ub,revealing the molecular mechanism of PINK1 catalyzing Ub.Furthermore,PP2 A was found to be involved in the regulation of PINK1-Parkin-mediated mitophagy pathway.Finally,the dynamic structure of K48-linked polyubiquitin was studied by combining fluorescence polarization and single-molecule fluorescence resonance energy transfer technology,and it was proved that the substrate protein regulates the dynamic structure of K48-linked polyubiquitin.