The Design And Application Of A Fluorescent PINK1 Biosensor

Ubiquitin exists in a wide range of eukaryotes and is highly conservative during evolution.Ubiquitin is also an important signalling protein,with ubiquination being one of the most important post-translational modifications.Also,ubiquitin itself undergoes post-translational modifications,such as phosphorylation and acylation.PINK1(PTEN-induced putative kinase 1)has been identified as the kinase of serine 65 of ubiquitin.PINK1 has multiple sub-cellular localizations and functions,among which its role has been demonstrated crucial in mitophagy.By sensing the loss of membrane potential in damaged mitochondria,PINK1 accumulates on the outer membrane of mitochondia and phosphorylates ubiquitin chains attached to proteins on the outer membrane of mitochondia and the UBL(ubiquitin-like domain)of Parkin,whose E3 ligase activity is initiated,inducing the recruitment of downstream mitophagic adaptors and robust mitophagy.Apart from mitochondrial localization,PINK1 also localizes to nucleus,cytosole,and mitochondrial plasma,whose functions remain elusive.Now,the research on PINK1 on the cellular level usually implements immunofluorescence stainning,western-blot and adding fluorescent protein tags.However,these techniques either damage the samples,are unable to mearsure in living cells and unable to mearure PINK1 activity,or change the natural state of PINK1.So it is urgent to develope a novel technique to monitor the distribution and activation of PINK1 in physiological or phathological conditions.cpFP(circular permutated fluorescent proteins)-based biosensors are widely used in sensing a variety of biological processes.cpFPs are highly sensitive to conformational change,with the conformational change of subnits transferring to cpFP and subsequently incudcing fluorescence change.Our prior research showed that,upon phosphorylation by PINK1,pUb undergoes conformational change between two states,namely the relaxed stated and the retracted state.In physiological condition,the ratio of the two conformation is close to 1:1.This project implements the conformational change of pUb and the sentitivity to conformation of cpFP to design a fluorescent PINK1 biosensor.By in silico and in vitro screening,we identified several constructs that respond to PINK1 phosphorylation.We charaterized the working mechanism of this biosenor by NMR,fluorescent spectroscopy and UVVis spectroscopy and demostrated its effecacy by measuring its response to mitochodrial damage in vivo.The designed sensor is structurally novel,expanding the design strategy of cpFP-based biosensor,and would be valuable for cell biology research related to PINK1 and the screening of drugs targeting the neurodegenarating disease linked to mitophagy disfunctions.