Effect Of RNF126 On Ubiquitination Modification,Degradation And Cytotoxicity Of Mutant Huntingtin

Huntington’s disease(HD)is an autosomal dominant neurodegenerative disease.Clinically,the patients are mainly manifested as progressive motor,cognitive and mental disorders.Pathologically,the patient is characterized by selective loss of projection neurons in the new striatum and neurons in the deep cerebral cortex.HD is caused by mutations in the HD gene,which encodes the Huntingtin(Htt)protein.Mutant HD gene encodes abnormally amplified polyglutamine fragment(Poly Q)at the amino terminal of protein,forming mutant huntingtin(m Htt).m Htt can exist freely(soluble m Htt)or misfold in cytoplasm and nucleus to form dense m Htt aggregates(insoluble m Htt).Both types of m Htt are cytotoxic and lead to cell damage and apoptosis through a variety of pathways,which is the root cause of the neuropathological changes causing HD.Therefore,inhibiting the abnormal accumulation and aggregation of m Htt,and/or promoting the degradation and clearance of m Htt,is of great significance in inhibiting the neuropathological changes of HD.In eukaryotic cells,the removal of misfolded proteins is mainly achieved through the autophagy lysosomal pathway(ALP)and the ubiquitin proteasome pathway(UPP).UPP is considered to be the most effective way to degrate m Htt.The degradation of staggered proteins by UPP is premised on their polyubiquitination,which is catalyzed by a wide variety of Ubiquitin ligase E3.Different types of E3 ubiquitin ligases have specificity in substrate recognition,so finding an E3 ubiquitin ligase that can effectively modify m Htt and promote its degradation by UPP has become a research hotspot in the treatment of HD.Ring finger protein 126(RNF126),a newly identified E3 ubiquitin ligase,belongs to the RING-finger domain family.RNF126 catalyzed polyubiquitination of specific substrates through its carboxy-terminal RING-finger domain and amino-terminal Znfinger domain,thus promoting UPP degradation of substrates.Recent studies have found that RNF126 is closely related to the degradation of pathogenic proteins in neurodegenerative diseases.In order to clarify whether RNF126 is related to the degradation of m Htt,this study analyzed the correlation between RNF126 and m Htt from the perspective of aggregation formation,protein degradation,cytotoxicity and ubiquitination modification by using a variety of cellular and molecular biological experimental methods.From the perspective of negative regulation,the calpaindependent cleavage effect of m Htt on RNF126 was preliminary revealed.1.RNF126 promoted the degradation of mutant huntingtin through ubiquitin proteasome and non-ubiquitin proteasome pathways and inhibited the cytotoxicity of mutant huntingtinTo determine whether the E3 ubiquitin ligase RNF126 is involved in the degradation of m Htt by UPP and inhibiting the cytotoxicity of m Htt,the effects of overexpression of RNF126 and small molecule interfering RNA(si-RNA)silencing RNF126 on the level of mutant Htt were detected.Fluorescence microscopy and Western blot analysis showed that overexpression of RNF126 could significantly reduce the levels of insoluble and soluble m Htt.Silencing RNF126 significantly increased the protein levels of both insoluble and soluble m Htt.Overexpression or silencing of RNF126 had no significant effect on the protein level of the co-transfected normal Htt.After blocking UPP with proteasome inhibitor MG132,the effect of overexpression of RNF126 on the reduction of soluble m Htt was inhibited,but the formation of m Htt aggregates was still effectively inhibited.Cell viability assay showed that overexpression of RNF126 could effectively inhibit m Htt-induced cell death.In contrast,silencing RNF126 significantly increased the cytotoxicity of m Htt.These results indicate that RNF126 can inhibit the cytotoxic effect of m Htt by promoting the degradation of m Htt.The degradation of soluble m Htt was dependent on UPP,while the reduction of insoluble m Htt was non-UPP dependent.2.RNF126 interacted abnormally with mutant huntingtinIn order to clarify whether the reduction effect of RNF126 on m Htt was based on the mutual binding,we analyzed the spatial localization and binding relationship between RNF126 and m Htt aggregates.The results showed that endogenous RNF126co-localized with m Htt aggregates.Laser scanning confocal microscopy(LSCM)tomography showed RNF126 was distributed in and around the m Htt aggregates..The immunocoprecipitation method(CO-IP)showed that RNF126 could bind to both normal Htt and mutant Htt,but the binding force was stronger with m Htt.3.RNF126 promoted the ubiquitination modification of mutant huntinhtinTo determine whether RNF126 promoted the degradation of m Htt by increasing its ubiquitination,we examined the effects of different RNF126 expression levels and RNF126 mutation [RNF126(C229/232A)] on the polyubiquitination and degradation of m Htt.Western blot analysis showed that overexpression of wild-type RNF126 significantly enhanced the polyubiquitination of m Htt and promoted its degradation.The effect of over-expression mutant RNF126 on enhancement of m Htt polyubiquitination modification and degradation of m Htt was significantly decreased compared with over-expression wild-type RNF126.Silencing RNF126 significantly reduced the polyubiquitination level of m Htt.Therefore,the polyubiquitination modification of m Htt by RNF126 is the cause of the degradation of soluble m Htt.4.RNF126 directly catalyzed the ubiquitination of mutant huntingtinIn order to determine whether RNF126 directly catalyzed polyubiquitin modification of m Htt,we induced and purified wild-type RNF126,functionally deactivated RNF126(RNF126 mutation)and m Htt in vitro.The three proteins were used as enzymes(wild-type RNF126,functionally deactivated RNF126)and substrates(m Htt),respectively.The results showed that wild-type RNF126 could directly catalyse the ligation of polyubiquitin chain to m Htt in vitro.The functionally deactivated RNF126 mutation lost its ability to catalyse the ubiquitination of m Htt.m Htt is a new catalytic substrate of RNF126,which is directly modified by polyubiquitination of m Htt by E3 ubiquitin ligase.5.Mutant huntingtin increased cleavage of RNF126In order to determine whether m Htt had a negative effect on RNF126 expression,we detected RNF126 m RNA and protein levels in N2 a cells transfected with normal and mutant Htt,and found that overexpression of m Htt had no effect on the transcription of RNF126,but significantly reduced its protein level.The level of GFP-RNF126 fusion protein was detected by anti-GFP antibody,and it was found that m Htt significantly reduced the level of GFP-RNF126,and there were obvious protease cleavage bands under the main band of GFP-RNF126.In conclusion,m Htt increases the protease cleavage of RNF126,leading to a decrease of RNF126 protein levels.Conclusion: In this study,we demonstrate for the first time that m Htt is a catalytic substrate for E3 ubiquitin ligase RNF126.RNF126 can directly act on m Htt,promote its polyubiquitination,promote the UPP degradation of soluble m Htt and nonUPP degradation of insoluble m Htt,and thus reduce the cytotoxicity of m Htt.We also found that m Htt down-regulated RNF126 levels by promoting RNF126 cleavage.RNF126 promotes the degradation of m Htt by promoting the polyubiquitination modification of m Htt,thus inhibiting its cytotoxic function.RNF126 provides a new target for the exploration of the treatment of HD from the perspective of regulating the function of UPP.The abnormal promoting effect of m Htt on the digestion and degradation of RNF126 provides a new scientific basis for further revealing the mechanism of the neuropathological changes of HD caused by m Htt through the impairment of UPP function.