Herp Promotes Proteasomal Degradation Of Mutated Huntingtin Through Enhancing Ubiquitination

Homocysteine-induced endoplasmic reticulum protein(Herp)is a stress response protein and encode by Homocysteine-inducible,ER-stress-inducible,ubiquitin-like domain member 1(Herpud1),with an ubiquitin-like domain in N-terminal.Herp is strongly up-regulated in response to early endoplasmic reticulum stress(ER stress),which promotes the clearance of misfolded protein.The phenomena of ER stress triggered by gathered misfolded proteins were found in many neurodegenerative diseases,such as Alzheimer's disease(AD),Parkinson's disease(PD)and Amyotrophic lateral sclerosis(ALS).It has been shown that Herp expression was up-regulation in AD and PD,and plays different function.In AD,Herp can enhance presenilin-mediated generation of amyloid beta-protein.And in PD,Herp protected dopaminergic neurons to promote the clearance of CCAAT enhancer-binding protein homologous protein through ubiquitin-proteasome pathways to PD cytotoxicity.Huntington's disease(HD)is an autosomal dominant neurodegenerative disease,the clinical character is progressive motor dysfunction,psychiatric disturbance and cognitive defects.Huntington's disease is caused by mutant huntingtin gene.The abnormality increased CAG repeats in exon1 huntingtin generates mutant Htt containing polyglutamine(Poly Q)expansion stretch.Mutant Htt is misfolded and forms soluble and insoluble Htt in the neuronal cytoplasm and nucleus.The soluble and insoluble mutant Htt are both toxic to cells,so inhibition of mutant Htt formation and promoting clearance of mutant Htt is great important to inhibit neuropathologic process of HD.In huntington's disease,the mutant huntingtin in the cell also can trigger the excessive ER stress response as well.The excessive endoplasmic reticulum stress is harmful to cells and cause cell apoptosis.While some ER membrane proteins are upregulated and promote the clearance of misfolded proteins,which is benefical to cell survival.Previous studies have shown that mutant Htt induced ER stress and leaded to increased Herp m RNA levels,but the role of its upregulation was not clear.So we detect the influence of mutant Htt on Herp expression using HD transgenic mouse and transfected mutant Htt N2 a cells,then analysed the degradation and cytotoxicity of mutant Htt at the different expression of Herp.Mutant Htt upregulated Herp protein level From the previous study,we know Herp m RNA expression is significantly increased in ER stress caused by mutant Htt,but it is not clear the protein level change,so we first detected the Herp protein levels in HD.The Herp protein was more expressed in R6/2 mouse brain than wild type at age of 5 week,8 week and 12 week from western blot results.And in Neuro 2a cell,it was transfected with normal Htt(Htt20Q-GFP)or mutant Htt(Htt160Q-GFP).The expression of Herp was examined by western blot in different times after transfection.The results show that Herp protein levels were higher in mutant Htt expressed cell compared with that in normal Htt express cell.Immunohistochemical results showed immunoreactivity of Herp significantly enhanced in cerebral cortex,striatum and hippocampus brain regions of R6/2 mouse.Those results show that mutant Htt could significantly increase Herp protein levels.Herp interacted with mutant Htt Mutant Htt could significantly increase Herp protein levels,we want to know the meaning of this phenomenon.We detected the intereaction between mutant Htt and Herp through double immunofluorescence technique and co-immunoprecipitation method.The Neuro 2a cell was co-transfected with Htt160Q-GFP and Cherry-Herp.The results showed that herp was diffusibly expressed in cytoplasm with soluble mutant Htt cell.And in the cell that expressed insoluble mutant Htt,the aggregate was encapsulated by strong positive granular of Herp.In the cell was separate transfected with Htt160Q-GFP,endogenous Herp also showed similar characteristics.Next we detected the interaction between Herp and Htt by protein immunoprecipitation method.The results showed that both Htt20 Q and Htt160 Q were co-immunoprecipitated with Herp.While there was more endogenous Herp was co-immunoprecipitation by Htt160 Q.These results suggest that both the normal Htt and mutant Htt can interact with Herp,but Herp can selectively interact with mutant Htt in HD.Herp promoted degradation of mutant Htt and suppressed Caspase-3 activation To further investigate the meaning of Herp upregulated and the interaction with Htt,we detected the impact of different expression of Herp on Htt.Under the fluorescence microscope,we observed that Herp overexpression could reduce the fluorescence intensity of mutant Htt(Htt160Q-GFP)and decrease the cell number with insoluble mutant Htt,while knock-down Herp could enhance the fluorescence intensity of mutant Htt and increase the cell number with insoluble mutant Htt.Meanwhile transfected normal Htt(Htt20Q-GFP)intracellular fluorescence intensity was significantly enhanced after Herp silence;whereas under the case of over expression of Herp,cells transfected with normal Htt fluorescence intensity only slightly weakened.The western blot result was consistent with the GFP fluorescence intensity changing.Overexpression of Herp significant decreased the protein level of insoluble and soluble Htt,and knock-down of endogenous Herp significant increased the levels of aggregated and soluble mutant Htt.The normal Htt protein level was also reduced but there was no significance between two groups after Herp overexpression,However,the normal Htt level was significant increased after knock-down of Herp.Moreover,over expression of Herp could reduce activity Caspase-3 protein level.Knock-down Herp increased the protein level of activity Caspase-3,and enhanced cell sensitivity to cytotoxicity of mutant Htt.From the RT-PCR the m RNA level of over expression plasmid was not changed after overexpression or knock-down Herp.The results showed that upregulated Herp enhanced the degradation of mutant Htt and reduced its cytotoxicity.Overexpression of Herp had no influence on normal Htt,suggesting that Herp selectively promoted the degradation of mutant Htt.Herp knock-down increased normal Htt indicated that physiologic level of Herp was necessary for maintaining the normal protein physiologic level of cells.Herp enhanced the degradation of soluble mutant Htt through peoteasome pathway To identify the degradation pathway of mutant Htt enhanced by Herp,we respectively blocked the two important protein degradation pathway in cells that was: the ubiquitin-proteasome pathway and autophagy lysosomal pathway.The results show that inhibition of the autophagy pathway had no obvious impact on the soluble and insoluble mutant Htt protein levels.And the proteasome inhibitor MG132 could significantly restore the degradation of soluble mutant Htt cause by Herp.Herp promoted ubiquitination of mutant huntingtin Herp promoted soluble mutant huntingtin degradation by the proteasome pathway,and the ubiquitin was an important degradation marker of proteasome pathways.In order to know whether Herp changed the ubiquitination of soluble mutant huntingtin,we observed the effect of Htt160 Q ubiquitination by different expression levels of Herp.The results showed that over expression of Herp enhanced the Htt160 Q ubiquitination level,while knock-down Herp attenuated Htt160 Q ubiquitination levels.The results showed that Herp can enhance mutant Htt ubiquitination and promoted its degradation by the ubiquitin-proteasome pathway.Ubquitin-like was a key domain for Herp suppress abnormal accumulation of mutant Htt and promote degradation of mutant Htt The two inhibitors were not able to block the effects of Herp on insoluble mutant Htt,which indicated that another way might take part in the effect of Herp on aggregated mutant Htt.In eukaryotic cells,the molecular chaperones inhibit the aggregatation of mutant Htt.The protein with ubiquitin-like domain or ubiquitin-like protein function could played a role as molecular chaperones in regulating the assembly substrate.To prove whether Herp with UBL play an chaperone-like role to inhibiting the formation of aggregates of mutant Htt,we constructed UBL domain deletion plasmid,HA-Herp-? UBL.We found that compared with HA-Herp,the effect of HA-Herp-?UBL on insoluble mutant Htt was slightly weakened,while on the insoluble mutant Htt,it was significantly weakened.That indicated that Herp played the role as a similar molecular chaperone to inhibit the formation of Htt aggregates and reduce the insoluble mutant Htt level.And the UBL was key domain for Herp suppressing abnormal accumulation of mutant Htt and promoting degradation of mutant Htt.Conclusion: This study found that mutant Htt upregulated Herp protein level and interacted with it.The upregulated Herp promoted mutant Htt degradation via the proteasome pathway by enhancing the ubquitination of mutant Htt and suppressed abnormal accumulation of mutant Htt,which decreased the level of mutant Htt and reduces cytotoxic of mutant Htt.