Polyubiquitin Chain-induced P62 Phase Separation Drives Autophagic Cargo Segregation

Autophagy is a highly conserved biological process in eukaryotes,which functions in protein turnover and homeostasis maintenance by cellular recycling of multiple cytoplasmic components including protein aggregates and damaged or superfluous organelles.Autophagy was thought to be a random-encapsulated,nonselective process for a long time.But recent years,the discovery of and characterization of LC3 family proteins and a variety of autophagy receptors has accelerated our understanding of selective autophagy.Different targets such as mitochondria,endoplasmic reticulum,lysosomes and protein aggregates are targeted by different receptor proteins for lysosome degradation through interaction with LC3.p62 was the first selective autophagy receptor described in mammalian cells and has been well studied for its role in protein aggregate clearance(aggrephagy).It can bind ubiquitin via UBA domain and LC3 via LIR motif,by which can target ubiquitinated cargo for autophagy degradation.With the ability to bind multiple interactors p62 was characterized as s scaffold protein acting in several important signaling pathways.p62 was accumulate in cytosolic inclusions in a number of human diseases and can form protein bodies in the cell called p62 body.We found that p62 body is a protein droplet with liquid properties rather than a protein aggregate,which is formed through liquid-liquid phase separation with the induction of polyubiquitin chain.This process is depended on polymerization via PB1 domain,binding ubiquitin through UBA domain,and the valency of polyubiquitin chain.Besides,we found that the phosphorylation of S403 site can promote p62 phase separation.And p62 body formation has a direct correlation with its degradation through autophagy.In addition to p62 and ubiquitin,LC3 can uniformly distributed in p62 in the form of lc3-? and can diffuse quickly inside p62 body.Besides,a variety of p62 interactors,such as NBR1,PRKCZ,MAP2K5 and Keap1 are all accumulated in p62 body and have varying degrees of influence on the mobility of p62 body.PDB is chronic bone disease which is characterized by focal areas of aberrant and excessive bone turnover,specifically increased bone resorption and disorganized bone formation.We found that the PDB related p62 protein mutations inhibit the formation of p62 body.This suggests that the failure of p62 body formation may be the pathogenesis of PDB.In conclusion,we found that polyubiquitin chain induced p62 phase separation drives the formation of p62 body and autophagic cargo segregation.This is the first time that the concept of phase separation is put forward in the autophagy field,which guides the research of other membrane-less compartment in autophagy field and implies the formation mechanism of cytoplasmic inclusion bodies in a variety of human disease and pathogenesis of PDB and thus provide a better theoretical basis and direction of the target of autophagy in the treatment of these human diseases.