Atypical Ubiquitination Of Mutant Huntingtin By WWP1Inhibits Degradation Of Mutant Huntingtin By Ubiquitin-proteasomal Pathway

Huntington’s diseases (HD) is an autosomal dominant hereditary neurodegenerative Disease, caused by abnormal CAG repeat (>35) in exon1of huntingtin gene. Mutant HD genes generates mutant Htt with a polyglutamine (poly Q) expansion at amino terminal, which forms insoluble aggregates in cell, and leads to cytotoxicity. Mutant Htt plays an important role in the pathogenesis of HD.The misfolded proteins in cells are mainly degraded by Ubiquitin-proteasome system (UPS). In the process of UPS degradation, misfolded proteins need to be ubiquitinated at first, which then be recognized by ubiquitin ligase E3. Ubiquitin ligase E3mediates activated ubiquitin transfers from ubiquitin conjugating enzyme E2to the lysine (Lys) residues of misfolded protein as a substrate. So ubiquitin ligase E3is the key enzymes of UPS. The lysine residues on the substrate protein could be polyubiquitination modified. And Ubiquitin has seven lysine residues as well, which are K6, K11, K27, K33, K48and K63. Polyubiquitination is a ubiquitin chain in end-to-end formation through one or more connections. In the seven kinds of ubiquitin connection, the most common connection is Lys-48and Lys-63linked chains. Lys-48linked chains are the typical ubiquitin modification, mediating UPS degradation. While other sites of lysine linked chains are atypical ubiquitin modification that do not only mediated UPS degradation, but also inhibit the typical ubiquitin modification degradation mediated by UPS. Lys-63linked chains are the most common of atypical ubiquitin modification. Thousands of protein ubiquitination processes are going on in cell, and this kind of specific degradation mechanism to a particular protein is mainly decided by ubiquitin ligase E3.Ubiquitin ligase E3mainly has HECT (homologous to E6AP C terminus) structure domain family, the structure of the RING-finger domain family and U-box structure domain family. HECT ubiquitin ligase E3extends ubiquitin polymer chain by itself. According to different N-terminal amino acid sequence, the HECT family is divided into three subfamilies: subfamily containing RLDs (RCC1-like domains) structure, subfamily containing the WW domain structure (Nedd4/as nedd4-1appears like E3) and subfamily not containing RLDs and WW domain structure (SI-HECT E3). WW domain-containing E3ubiquitin protein ligase1(WWP1) is one ubiquitin ligase E3of the HECT structure domain family. It consists of C2-WW-HECT structure domain. The four WW domains in the middle of structure identify proline-rich region (PRR), which determine the substrate specificity. And C terminal of the HECT structure domain determines the connection form as the active area, such as different forms of ubiquitin on different substrates including single ubiquitination, or Lys-48and Lys-63linked chains polyubiquitination form.WWP1mediates polyubiquitination modification to a variety of pathogenic protein in neurodegenerative diseases. WWP1interacts with Poly Q sequence in atrophin-1by WW structure domain and polyubiquitination modifies atrophin-1in poly Q diseases, such as dentatorubral-pallidoluysianatrophy (DRPLA). At the amino terminal of Htt, there are both PRR and Poly Q sequence. Therefore, WWP1may interact and ubiquitination modify Htt through the WW domain structure and PRR and/or Poly Q sequence. Mutant Htt modulates the expression of multiple genes at transcription level, such as UBR1, UBE3B ubiquitin ligase E3, which may also affect the expression of WWP1, and thus affect its own ubiquitin modification and degradation leading to relevant pathological damage.There is no report show that whether WWP1could modify mutant Htt as an E3ligase and WWP1 expression in HD. In order to prove the above hypothesis, we studied the influence of WWP1to mutant Htt.WWP1was collocalized with aggregates of mutant huntingtin To analyze the possibility of interaction between WWP1and mutant Htt, we first observed the localization relationship between endogenous and transfected WWP1and mutant Htt in N2a cells expressing mutant Htt. Immunofluorescence staining data showed that the endogenous WWP1and Htt20Q were both dispersed in the cytoplasm in N2a cells transfected with normal Htt (Htt20Q); while WWPlwas recruited in part of the Htt aggregates in N2a cells transfected with mutant Htt (Httl60Q). Moreover, recruited WWP1was co-localized with partial mutant Htt aggregates in N2a cells co-transfected with Htt160Q and WWP1. After analyzing the brain tissue of HD transgenic mice R6/2with filtration traps, we found that WWP1could combine with mutant Htt aggregates. These data indicate that WWP1could interact with mutant HttWWP1promoted ubiquitination of mutant huntingtin To clarify whether WWP1could modify mutant Htt as an E3ligase, firstly, we overexpressed or silenced WWP1in N2a cells expressing with mutant Htt. By immunoprecipitation analysis, we found that overexpression of WWP1could increase mutant Htt ubiquitination, and silence WWP1could reduce mutant Htt ubiquitination when proteasome degradation pathway was inhibited. While WWP1-mediated ubiquitination of mutant Htt was disabled when the active site of WWPl(cysteine was mutated to serine in the886site of WWP1) was mutant. It is suggested that WWP1could modify mutant Htt by ubiquitination.WWP1elevated the amount of mutant huntingtin aggregates and enhenced its cytotoxicity To investigate the role of WWP1-mediated ubiquitination of mutation Htt in the UPS pathway, we tested the effect of WWP1on the stability of mutant Htt. Western blot analysis showed that overexpression of WWP1could significantly increase the expression of aggregated and non-aggregated mutant Htt in N2a cells expressing mutant Htt. While silent WWP1had the opposite effect. By counting the aggregates under a fluorescence microscope, we found that overexpression of WWP1significantly increased the recruitment of aggregates and silent WWP1reduced the recruitment of aggregates. Filtration trap analysis showed that overexpression of WWP1significantly elevated mutant Htt aggregates, while silence WWPlwas the opposite. RT-PCR analysis showed that neither overexpression of WWP1nor silence WWP1had effect on the expression of mutant Htt, Thus, WWP1-mediated ubiquitination modification of mutant enhanced the stability of mutant Htt. Trypan blue staining indicated increased expression of WWP1significantly increased the cytotoxic of N2a cells expressing mutant Htt while decreased WWP1significantly inhibited the cytotoxic.WWP1inhibited degradation of mutant huntingtin by ubiquitin-proteasomal pathway To clarify whether the enhancement of WWP1-mediated ubiquitination modification on mutant Htt stability was caused by the inhibition of proteasome degradation pathway, we observed the effect of inhibition of proteasome activity on the enhanced degradation of mutant Htt by silencing WWP1. By using Chlorhexidine Dihydrochloride (CHX) to block protein synthesis at different time point, we investigated the effect of WWP1on the degradation of mutant Htt in N2a cells expressing mutant Htt. Western blot analysis showed that silence WWP1could promote mutant Htt degradation, the longer, the greater degradation. After treatment with proteasome inhibitor MG132, the effect of silent WWP1on mutant Htt degradation was blocked. All these data showed that WWP1-mediated ubiquitination modification inhibited mutant Htt proteasome degradation pathway.WWP1ubiquitinated mutant huntingtin in way of Lys-63linkages Given that WWP1-mediated ubiquitination modification inhibited mutant Htt ubiquitin-proteasome degradation pathway, WWP1may modify mutant Htt in Atypical ubiquitination way. To prove this hypothesis, we first constructed seven mutant ubiquitin plasmids according to seven different Lys sites. Then we investigated the effect of different mutant ubiquitin on WWP1-mediated ubiquitination in mutant Htt expressing N2a cells. Immunoprecipitation analysis showed that overexpression of wild-type WWP1increased the level of WWP1-mediated mutant Htt ubiquitination, while the ubiquitination level in cells transfected with ubiquitin63Lys mutant plasmid was significantly lower than that in cells over-expression wild-type WWP1, which was similar as in the cells not over-expression wild-type WWP1; In the other six mutant Lys plasmids transfecting cells, the mutant Htt ubiquitination level was similar as that in wild-type WWP1overexpression cells. The data indicated that WWP1-mediated ubiquitination developed Lys-63linkage poly-ubiquitin chain to modify mutant Htt in atypical ubiquitination way, which could not mediate mutant Htt to be recognized and degradated by the proteasome.Mutant huntingtin upregulated expression of WWP1Mutant Htt can influence the expression of multiple genes, including E3ubiquitin ligase gene, at the transcription level. In order to clarify whether mutant Htt could influence UPS on their own degradation by affecting WWP1expression, We tasted WWP1expression level in R6/2mice and N2a cells expressing mutant Htt. The results showed that the mutant Htt could upregulate WWP1expression at the transcriptional level. In R6/2mouse brain, WWP1protein level was significantly increased compared with wild-type mouse; WWP1protein level was not obviously changed in Htt160Q cells compared with that in Htt20Q cells48h after transfection, while WWP1protein level was significantly increased in Htt160Q cells72h after transfection. RT-PCR data showed that the mRNA level of WWP1in Htt160Q cells was almost the same as that in Htt20Q cells48h after transfection, but the mRNA level of WWP1was unregulated in Htt160Q cells72h after transfection.Conclusions:Ubiquitin ligase WWP1could atypical ubiquitination mHTT and inhibit its degradation in way of Lys-63linkages,inducing cytotoxicity.The cytotoxicity of mHtt probably related with the accumulation of the mHtt,due to the inhibiton of degradation of Ubiquitin-proteasome system,which related with the up-expression of WWP1and enhancement of atypical ubiquitination.WWP1could to be the regulator of mHtt degradation and target of HD therapy.