Pin1 Regulates The Monoubiquitination And Activity Of CHIP Via UbE2W

In the natural situations,cells are regularly exposed to various stress conditions that may lead to protein misfolding,but also in the absence of stress,misfolding proteins occur during protein synthesis.Usually misfolded protein cannot reach a functional conformation and form protein aggregates,which has a fatal effect on the cell.The central components of the protein quality control system(PQCS)are as follows: chaperones,which promote protein folding and refolding,and degradation machineries,namely the ubiquitin-proteasome and autophagy.Carboxy-terminus of the heat shock protein 70-interacting protein(CHIP)has dual functions,one as a cochaperone,and the other as an E3-ubiquitin ligase.CHIP interacts with hsp70 through its amino terminus to help unfolded protein folding and misfolded proteins refolding.CHIP regulates the ubiquitin proteasome system(UPS)degradation of damaged proteins to prevent cellular toxicity.CHIP appears to play a central role in the degradation of neurodegenerative disease-related aggregated proteins.CHIP is a neuroprotective protein and it can facilitate the aggregated protein clearance by UPS.Recent studies have found that the monoubiquitination of CHIP is necessary for its E3 ubiquitin ligase activity.Ub E2 W,an E2 ubiquitin-conjugating enzyme,proved to promote monoubiquitination of the sole lysine residue K2 on CHIP.However,the mechanism by which the protein-protein interactions between CHIP and its substrates,and CHIP with Ub E2 W regulates its activity,remains unknown.Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1(Pin1)belongs to the family of the peptidyl-prolyl cis/trans isomerase.Pin1 specifically recognizes and catalyzes the phosphorylated Serine/Threonine-Proline(Ser/Thr-Pro,S/T-Pro)motifs cis/trans isomerization of its substrates.Pin1 possesses two major domains,namely the WW domain and the PPIase domain.Two domains can both bind to the phosphorylated Ser/Thr-Pro motif independently.This allows Pin1 to exert its molecular function as an isomerase via its PPIase domain,leading to the cis/trans conversion of its substrate to elicit the intended biological outcomes.There are four Ser/Thr-Pro motifs in CHIP,S19-Pro,S23-Pro,T246-Pro and S273-Pro,and three of them have been proved to be phosphorylated.Since phosphorylated Ser/Thr-Pro motif is known to be a target for Pin1,we determined CHIP may be a substrate of Pin.In our previous work,we found that Pin1 could interact with CHIP and enhanced activity of CHIP.All these data strongly suggest that CHIP may be a candidate substrate of Pin1.Whether Pin1 catalyzes the cis/trans isomerization of S/T-Pro motifs in CHIP or not,and how the modification and activity of CHIP is regulated by Pin1,however,remains largely unknown.The occurrence of numerous diseases that caused by mutant protein or misfolded protein is related to the dysfunction or damaged activity of Pin1,E2 and /or E3 of UPS,and chaperone.And then,the function of PQCS is disordered,which leads to the mutant protein or misfolded protein cannot be degraded and cleared in time.CHIP promotes the degradation of spinocerebellar ataxia type 3(SCA3)mutant ataxin 3 through UPS,and this function can be significantly up regulated by Pin1.However,it remains unknown about whether mutant ataxin 3 impairs the functional activities of Pin1,E2 or E3,also whether mutant ataxin 3 inhibits its degradation through UPS via impairing the functional activities of Pin1,E2 or E3.Here we used nuclear magnetic resonance(NMR)to detect possible binding sites of CHIP with Pin1,as well as cis/trans isomerization.We show that the interactions among Pin1,CHIP,substrate of CHIP(ataxin 3-80Q)and Ub E2 W,as well as the effect of CHIP activity by its substrate.From the perspective of protein-protein interaction,the role and molecular mechanism of Pin1 regulating CHIP activity were further analyzed in this study.PPIase domain of Pin1 interacts with S273-Pro motif in CHIPIn order to determine whether CHIP interacts with Pin1 and to localize the binding sites,we first detected the endogenous interaction between CHIP and Pin1 in mouse brain or mouse N2 a cells by coimmunoprecipitation(Co-IP)assay.The results showed that CHIP interacted with Pin1.Subsequently,we sought to determine which domain of Pin1 and which site in CHIP were responsible for the binding between them.Co-IP assays were performed using deletion functional domains of Pin1 and CHIP site mutants.The results showed that CHIP interacted with Pin1 PPIase domain through its S273-Pro motif.The binding of Pin1 does not affect the cis/trans isomerization of CHIPIn order to determine whether the S273-Pro motif of CHIP directly binds to Pin1,three synthesized short peptides(20 amino acid residues),which containing S-Pro motifs of CHIP,with or without recombinant Pin1 were detected by 1D NMR-based titrations.It was found that significant chemical shift perturbation could be observed only for the S273-Pro peptide when added Pin1,but not for S19/23-Pro peptide or T246-Pro peptide.Next work was to clarify whether Pin1 catalyzed conformational change of the phosphorylated S273-Pro in CHIP,the phosphorylated short peptide(20amino acid residues)and U-box phosphorylated long peptide(63 amino acid residues)were synthesized and then the 1H-1H(2D)ROESY experiment was performed.We found that the p S273-Pro motif in short peptide or in the long peptide,containing Ubox functional domain,abled to cis/trans isomerism themselves efficiency,and the addition of Pin1 had no effect on this phenomenon.So,the binding of Pin1 does not affect the cis/trans proline isomerization of CHIP.Pin1 promotes CHIP itself binding and CHIP interaction with its substrateThe Co-IP results showed that overexpression of Pin1 significantly enhanced the binding between CHIP and itself,while interfered with Pin1 inhibited CHIP itselfbinding affinity.Immunofluorescence results showed that GFP-ataxin 3-80 Q,Pin1 and CHIP were well colocalized in the Pin1 overexpressed of both N2 a and HEK293 cells.Further,in stable expression of spinocerebellar ataxia type 3 mutant protein(ataxin 3-80Q)cell lines,we found that ataxin 3-80 Q interacted with CHIP and Pin1,upregulating of Pin1 increased the affinity of mutant ataxin 3 for CHIP,while interfered with Pin1 impaired the capacity for ataxin 3-80 Q and CHIP binding.Therefore,Pin1 promotes CHIP itself binding and CHIP interaction with its substrate.Pin1 enhances CHIP substrate degradation activity via promoting monoubiquitination of CHIPOur previous studies have found that Pin1 enhances CHIP substrate degradation activity,and promotes the degradation of ataxin 3-80 Q by CHIP.Monoubiquitination modification of three amino terminal lysine residues K2,K4,and K7 activate the E3 ubiquitin ligase activity of CHIP.Therefore,the interaction between Pin1 and CHIP may affected the level of CHIP monouniubiquitination.Western blot analysis showed that overexpression of Pin1 significantly enhanced the CHIP monouniubiquitination,while knockdown of Pin1 that was opposite.The K4 R or K7 R mutations did not significantly affect the CHIP monoubiquitination that was enhanced by Pin1,while the K2 R mutation significantly inhibited Pin1-dependent monoubiquitination of CHIP.Further experiments found that overexpression of Pin1 significantly promoted the polyubiquitination of wild-type CHIP and K4 R or K7 R mutants.However,polyubiquitination was not enhanced when Pin1 was overexpressed in cells containing the K2 R single-point mutant or the K2 R,K4R,and K7 R triple mutant simultaneously(K2R+K4R+K7R).K2 R,K4R,or K7 R single-point mutations inhibited the ability of CHIP to degrade ataxin 3-80 Q,but the simultaneous mutation of all the three sites(K2R+K4R+K7R)had the greatest impact on CHIP functional activity and cell viability.Pin1 promotes the monoubiquitination of CHIP by enchancing the binding of Ub E2 W and CHIPUb E2 W is the only known ubiquitin-conjugating enzyme E2 that can efficiently monoubiquitinate the K2 site on CHIP.Pin1 has no ubiquitin-conjugating enzyme E2 activity but can enhance the monoubiquitination of K2 site on CHIP.These suggests that Pin1 may improve the monoubiquitination of CHIP through Ub E2 W.Indeed,our results showed that Pin1 increased the binding of Ub E2 W to CHIP and silencing of Ub E2 W impaired CHIP's ability to reduce the level of mutant ataxin 3.Co-IP results showed that the binding affinity between CHIP and Ub E2 W was significantly enhanced when Pin1 was overexpressed,whereas silencing Pin1 significantly weakened their affinity.Pin1 up-regulates the monoubiquitination of CHIP by interacting with Ub E2WProtein protein interactions are typically based on the association of specific structural regions within the proteins.Pin1 promotes the interaction between Ub E2 W and CHIP,thereby enhancing the monoubiquitination of CHIP by Ub E2 W.This suggests that Pin1 may also bind Ub E2 W.To assess this possibility,the localization of Pin1,Ub E2 W,CHIP and CHIP substrates(ataxin 3-80Q)was detected by immunofluorescence assay.Results showed that these four proteins were well colocalized in N2 a and HEK293 cells.The binding of endogenous Pin1 and Ub E2 W in N2 a cells was detected by Co-IP.PPIase domain of Pin1 interacted with Ub E2 W.Primary structural sequence analysis of the Ub E2 W protein showed that it contained two S-Pro motifs.Co-IP analysis showed that S68-Pro in Ub E2 W combined with the Pin1 PPIase domain,and this site may affect the monoubiquitination of CHIP.Mutant ataxin 3 reduces the monoubiquitination of CHIP by weakening the binding of Pin1 to Ub E2WAforementioned results suggest that Pin1 may play a role similar to that of adaptor protein.Pin1 appears to regulate the assembly and function of the Ub E2WCHIP-substrate complex.To see if the abnormal accumulation of mutant proteins affects the assembly and state of this complex,the level of monoubiquitination of CHIP was detected.Western blot results showed that the level of monoubiquitination of CHIP was high in the ataxin 3-20 Q cells,but was hardly detected in the ataxin 3-80 Q cells.IP with an anti-CHIP antibody showed that the binding capacity between CHIP and Ub E2 W was significantly reduced in the ataxin 3-80 Q group.IP with an anti-Pin1 antibody showed that in the ataxin 3-80 Q group,the binding capacity between CHIP and Ub E2 W was also weakened significantly.These results explains that mutant ataxin3 reduces the monoubiquitination of CHIP by weakening the binding of Pin1 to Ub E2 W.Conclusion: our study found that Pin1 regulates the E3-ubiquitin ligase function of CHIP in a non-peptidyl prolyl cis/trans isomerism manner.Pin1 promotes the interaction between Ub E2 W and CHIP,thereby enhancing monoubiquitination of the K2 site on CHIP,and mediates the formation of CHIP functional complex(including CHIP-CHIP,CHIP-specific substrate ataxin 3-80 Q,CHIP-CHIP's modification/regulatory factor Ub E2W),which supports its functional activity and protective effect of cell viability.Mutant protein substrate(ataxin 3-80Q)inhibits the binding of Pin1 and Ub E2 W and weaken the ubiquitination of CHIP by Ub E2 W,leading to ataxin 3-80 Q accumulation.