| The ubiquitin-proteasome system(UPS)is essential for maintaining the homeostasis of proteins in cells.The recognition of polyubiquitin chains by ubiquitin receptors on the proteasome is the first step for proteasome-mediated substrates degradation.Study on the specific recognition mechanism of ubiquitin receptors to ubiquitin chains is essential for better understanding the ubiquitin-proteasome system.Recognition between ubiquitin receptor Rpn13 and the K48-linked ubiquitin chain could promote the degradation of ubiquitinated substrates by the proteasome.However,only the complex structure of Rpn13NTD and ubiquitin monomer has been reported to data,how Rpn13 specifically recognizes the K48-linked ubiquitin chain is still unclear.Using single molecule fluorescence resonance energy transfer(smFRET),we found that K48-diUb showed three different conformations in solution.Rpn13NTD bound with one of these three conformations through conformational selection mechanism,and the binding mode was the same for those of the foll length Rpn13 and longer K48-linked ubiquitin chain.Using liquid NMR technology,we analyzed the complex structure between Rpn13NTD and K48-diUb,and it was found that Rpn13NTD simultaneously contacted with the distal and proximal subunits of K48-diUb,which was mutually corroborated by the results of smFRET.At the same time,we found that the R104E mutation of Rpn13 could significantly decrease its binding affinity for the K48-linked ubiquitin chain,leading to the accumulation of ubiquitinated substrates in cells.This study shows the interaction mechanism of the complex between Rpn13 and K48-linked Ubiquitin chain,it is able to help development of drug for the regulation of proteasome function.Ubiquitin(Ub)is a small signaling protein,and it can covalently modify other proteins,which is called ubiquitination.Besides,ubiquitin itself can also be phosphorylated,the Ser65 site of ubiquitin can be phosphorylated by PINK1.PINK1 is the currently known and most reported ubiquitin kinase.It is anchored on the outer membrane of the mitochondria through the N-terminal transmembrane region.PINK1 is cleaved by the PARL protease on the inner mitochondrial membrane,generating soluble form of PINK1(sPINK1).sPINK1 is a proteasomal substrate,and it can be degraded by the ubiquitin-proteasome system through the N-terminal degradation pathway.Therefore,the levels of PINK1 and sPINK1 in normal cells are very low.Under certain physiological and pathological conditions,the activity of the proteasome is decreased,and the reduced activity of the proteasome causes the increase of the level of sPINK1.Consequently,the undegraded sPINK1 increases the level of pUb in the cell.Phosphorylation can regulate the tertiary structure of Ub to produce a new conformation-retracted state.At the same time,we call the conformation similar to wild-type Ub as relaxed state.The ratio of relaxed and retracted conformations could be regulated by pH.PINK1 is not only a kinase for ubiquitin monomer,but also a kinase for polyubiquitin chains.Whether and how phosphorylation affects the structure and function of K48-linked ubiquitin chain is unclear.K48-linked ubiquitin chain is the most abundant in cells and is mainly used to mediate the degradation of ubiquitinated substrates by proteasome.K48-diUb is the shortest K48-linked ubiquitin chain and the basic unit of ubiquitin receptors recognition.K48-diUb has three different conformations in solution.After phosphorylation,each subunit of K48-diUb has two conformations,relaxed and retracted.Three inherent ubiquitin receptors(Rpn1,Rpn10 and Rpn13)on 26S proteasome selectively bind to specific conformations of K48-diUb through conformational selection.Using smFRET titration,we found that the ubiquitin receptor Rpn1/Rpn10 selected the open state of K48-diUb,while Rpn13 preferred the closed state of K48-diUb,and the conformational preference was not affected by ubiquitin phosphorylation;By NMR titration,we found that all three ubiquitin receptors preferentially bound to the relaxed state of phosphorylated K48-diUb.In addition,we also found that phosphorylation could change the tertiary structure of the subunits in K48-diUb,which would further influence the quaternary structure,Phosphorylation of K48-diUb dramatically decreased the population of Rpn1/Rpn10 preferred conformations,causing inhibition the binding of Rpn1/Rpn10 to K48-diUb.However,phosphorylation has no significant effect on the binding of Rpn13 to K48-diUb.Using TIRF technology to study the interaction between K48-linked ubiquitin chain and the ubiquitin receptors from the entire proteasome level,we found that phosphorylation could weaken the binding of K48-linked ubiquitin chain to the proteasome.The results of in vitro and in vivo experiments found that phosphorylation inhibited the degradation of ubiquitinated substrates by the proteasome.In addition,we also found that the increase of sPINK1 level could promote the formation of aggregate in cells and increase the cell death.In summary,our research is focused on the ubiquitin-proteasome system,the specific recognition of the K48-linked ubiquitin chain by ubiquitin receptors,and the regulation of the struture and function of the K48-linked ubiquitin chain by phosphorylation.Using smFRET,we found that the ubiquitin receptor Rpn13 specifically bound to the closed state of the K48-linked ubiquitin chain.At the same time,using liquid NMR,we analyzed the complex structure of Rpn13NTD and K48-diUb,indicating the binding mechanism between Rpn13 and the K48-linked ubiquitin chain.In addition,with combined use of NMR and smFRET technology,we found that phosphorylation could remodel the structure of the K48-linked ubiquitin chain at the tertiary structure and the quaternary structure level,and the structure change was able to affect the binding of the three ubiquitin receptors to K48-linked ubiquitin chain.Through TIRF experiments,we further confirmed that phosphorylation of ubiquitin could inhibit the binding between 26S proteasome and ubiquitinated substrate,and it finally affected the degradation of ubiquitinated substrates by the proteasome. |
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