ENC1 Promotes Degradation Of Mutant Huntingtin Hrough Ubiquitin-Proteasome System

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal expansion of CAG repeats (>35) in the first exon of HD gene encoding a protein called huntingtin (Htt). Mutant Htt has been found to misfold, forming intracellular insoluble aggregates in patients, transgenic animal models and cell models of HD. These aggregates are thought to play an important role in HD pathogenesis.Ubiquitin-proteasome system (UPS) is the major intracellular proteolytic enzyme system, which can clear the misfolded protein, protecting cells from the impairment of aggregates. In the process of UPS degrading misfolded proteins, the substrate proteins need to be ubiquitinated first. Ubiquintin ligase (E3) is the key enzyme for ubiquitination of substrates, which delivers activated ubiquitin molecule from ubiquitin conjugating enzyme E2 to substrates, conferring specificity to the process by selectively binding to a protein target. The way that E3 ligases mediates ubiquitin transfer to substrates divides E3 into two broad categories:HECT (homologous to E6AP C terminus) family and RING-finger family. RING-finger family is a functional complex composed of many proteins. Cullin-RTNG complex is the major member of RING-finger family, including three types of complex, SCF(Skpl cullin F-box, SCF), ECS (Elingin C-cu12 SOCS, ECS) and Cul3-based. Cul3-based is formed by RING protein, Cu13 and BTB-Kelch protein, the BTB-Kelch protein, as an adaptor, deciding the specificity of substrates.Ectodermal Neuronal Cortex 1 (ENC1), also named nuclear-restricted protein/brain (NRP/B), is a BTB-Kelch protein, which has a BTB domain in N-terminal and six Kelch repeat domains in C-terminal. Similar to BTB-Kelch proteins, ENC1 works a substrate-speicific adaptor to constitute a Cul3-based E3 complex, modulating the ubiquitination and degradation of substrates.Several kinds of E3 could recognize mutant Htt, mediating its degradation by UPS. Decrease in E3 expression leads to dysfunction in degradation of mutant Htt due to impairment of UPS function, which results in intracellular accumulation of mutant Htt. However, it has not reported whether ENC1 modulate the degradation of mutant Htt by UPS as an adaptor of Cul3-based E3.In this study, the effect on aggregation and cytotoxicity of mutant Htt by altered expression of ENC1 was first examined.Then, we analysed the interaction between ENC1 and mutant Htt, ubiquitiniation and degradation of mutant Htt by altered expression of ENC1, Finally, the influence of mutant huntingtin on expression of ENC1 was detected.ENC1 reduces the aggregation of mutant huntingtin. To identify that ENC1 can reduce aggregation of mutant Htt. Under fluorescence microscope, we observed that ENC1 overexpression could reduce the aggregation of mutant Htt and ENC1 silence could increase the aggregation of mutant Htt. It was confirmed by western blot that both insoluble Htt and soluble Htt was reduced by ENC1 overexpression, and it was reversible by ENC1 silence. At the same time, RT-PCR results showed that ENC1 overexpression or ENC1 silence did not alter the expression of mutant Htt. ENC1 could promote degradation of mutant Htt and reduce aggregation of mutant Htt.ENC1 inhibits cytotoxicity of mutant Htt. Whether ENC1 inhibited the cytotoxicity of mutant Htt by mediating the degradation of mutant Htt, we used flow cytometry and the activity of caspase-3 by Western blot to dectct the cytotoxicity of mutant Htt,which would be impacted by changed expression of ENC1. ENC1 or ENC1-SiRNA and 20Q-Htt/160Q-Htt were co-transfected into N2a cells and death cells were decteded by PI staining. By flow cytometry, the results showed that ENC1 overexpression didn't change the death rate of 20Q-Htt cells, but reduced 160Q-Htt cells significantly. However, when ENC1 was silenced, the cell death rates of 20Q-Htt and 160Q-Htt both increased. Morever, it was increased more significantly in 160Q-Htt cells. ENC1 overexpression could surpress the activation of caspase-3 induced by mutant Htt and ENC1-SiRNA could promote the activation of caspase-3. So ENC1 could inhibit the cytotoxicity induced by mutant Htt. ENC1 interacts with mutant Huntingtin. To know if ENC1 is associated with mutant Htt, the relationship of ENC1 to mutant Htt was observed by using immunofluorescent staining and co-immunoprecipitation. Immunofluorescent staining showed that, in N2a cells transfected with normal Htt (20Q-Htt), both transfected 20Q-Htt and endogenous ENC1 were diffusely expressed in the cytoplasm. In N2a cells transfected with mutant Htt (160Q-Htt), the transfected 160Q-Htt was found to forme aggregates. Interestingly, ENC1 was found to colocalize to mutant Htt aggregates. Co-immunoprecipitation analysis found that, the endogenous ENC1 was not precipitated by antibody against Htt in N2a cells expressing 20Q-Htt; however, in N2a cells transfected with 160Q-Htt, the endogenous ENC1 was found to be precipitated by antibody against HA-Htt, appearing an increase in molecular weight. Furthermore, when ENC1 was co-transfected with with 20Q-Htt or 160Q-Htt in N2a cells, only 160Q-Htt was precipitated by antibody against Myc-ENC1. The co-localization of ENC1 with mutant Htt aggregates and co-immunoprecipitation of ENC1 with mutant Htt indicates ENC1 interatc with mutant Htt. The increase in molecular weight of precipited ENC1 might be due to its self-ubiquitination.ENC1 promotes ubiquitination and degradation of mutant huntingtin. To clarify whether ENC1 associated with mutant Htt as an E3 liagse and promote ubiquitination of mutant Htt, we observed the effect of ubiquitination and degradation of mutant Htt by different expression levels of ENC1. The results show that ENC1 promote ubiquitination of total protein. Mutant Htt was immunopreciptated by anti-HA-Htt antibody. The ubiquitination of mutant Htt was stronger from cells cotransfected ENC1 with 160Q-Htt than vector and 160Q-Htt. Degradation of mutant Htt by ENC1 was blocked by proteasome inhibitor MG132. The ubiquitination of mutant Htt was weeker from cells cotransfected ENCl-SiRNA with 160Q-Htt than si-control with 160Q-Htt. Accumulation of mutant Htt by ENC1-SiRNA was enhanced by proteasome inhibitor MG132. So we can identify that ENC1 could promote ubiquitin of mutant huntingtin by interacting with mutant Htt.Mutant Huntingtin decreases expression of ENC1.Mutant Htt could affect a variety of genes expression. The expression of ENC1 was altered by mutant Htt using HD transgenic mouse model and HD cells models. By Western blot, we found that,the protein levels of ENC1 was lower in HD than in wild type, and the mRNA difference of ENC1 was the same as the protein. So, the expression of ENC1 was repressed in HD than in wild type.To further determinate whether mutant Htt inhibited the function of UPS by deregulating the expression of ENC1, the influence of ENC1 on mutant Htt was examined using HEK293 cell stably expression GFPu which contained the proteasome degradation signaling CL1. The Western Blot confirmed that the 160Q-Htt could significantly inhibit the degradation of GFPu, and ENC1 could reverse the phenomenon which was blocked by the proteasome inhibitor MG132.Conclusions:ENC1 could promote proteasomal degradation of mutant Htt through ubiquitining mutant Htt, therefore, inhibiting cytotoxicity of mutant Htt. Mutan Htt could impair the function of UPS through decreasing expression of ENC1.