The Effect On The Ubiquitination, Degradation, Inclusion Formation And Apoptosis Of Ataxin-3 Modified By SUMO-1

Background:The hereditary spinocerebellar ataxias(SCAs) are a heterogeneous group of neurodegenerative disorders.To date,at least 28 gene loci responsible for SCAs have been mapped,in which 18 pathogenic genes have been cloned.Among them,spinocerebellar ataxia type 3/Machado -Joseph disease(SCA3/MJD) is the most common subtype.The gene for SCA3/MJD has been cloned and designated as MJD1.SCA3/MJD is one of the polyglutamine(polyQ) diseases caused by an expansion of a polyQ stretch near the C-terminus of the MJD-1 gene product,ataxin-3.Up to now,the physiological function of ataxin-3 is unknown,and the pathogenesis of the expansion of a polyQ stretch near the C-terminus is still not well illuminated.Studies have found that post-translational modification of the disease proteins plays a critical role in their physiological function and pathogenesis.Ten years after its discovery,the small ubiquitin-like protein modifier(SUMO) has emerged as an important post-translational modification factor.SUMO family contains SUMO-1,SUMO-2, SUMO-3,SUMO-4 isoforms.Similar to ubiquitin,SUMO attachment to proteins which referred to as "SUMOylation",enters a multi-step enzymatic pathway.This reversible pathway provides a rapid and efficient way to modulate much prominent and basic function,such as subcellular localization,nuclear transport,transcriptional regulation,and protein stability.Similar to ubiquitin,SUMO are linked directly to the amino sidechains of lysine residues and,in some instances,both modifiers target the same substrate.This suggests a dynamic interplay between the related ubiquitination and SUMOylation pathways.SUMO immunoreactivity has been observed within inclusions in numerous neurodegenerative diseases including Alzheimer disease,multiple system atrophy,polyQ diseases(SCA1,SBMA,DRPLA,Huntingtin disease) and Parkinson's disease.The identification of huntingtin,ataxin-1,tau andα-synuclein as SUMO substrates further supports the involvement of SUMOylation in the pathogenesis of neurodegenerative diseases.We have found that the N-terminus of ataxin-3 interacted with SUMO-1 by yeast two-hybrid techniques screening human brain cDNA library,then confirmed the interaction in eukaryocyte by immunofluorescence-laser cofocalization and co-immunoprecipitation, and SUMO-1 modification might have toxic effect on polyQ-expanded ataxin-3.By online SUMOylation analysis protocol,we found that ataxin-3 contains SUMOylation motif(165VKGD168) at K166.Then by Ni-NTA precipitation and western-blot,we detected that K166 was the key amino acid of wild-type and polyQ-expanded ataxin-3 for SUMO-1 modification.We found that SUMO-1 modification didn't change the subcellular localization of wild-type and polyQ-expanded ataxin-3 using immunofluorescence initially.Our previous data indentified that ataxin-3 was the substrate of SUMO-1,and SUMOylation participated the pathogenesis of SCA3/MJD.Objective:To research the influence of SUMO-1 modification on the ubiquitination,protein degradation,formation of intranuclear inclusions, and apoptosis of wild-type and polyQ-expanded ataxin-3;And explore the effect on cytoplasmic/nuclear distribution further in eukaryocyte level.Methods:1.Recombinant DNA technology,Western-blot,GFP fluorescence technique were undertaken to construct and detect the expression of pEGFP-N1 eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3.2.Subcellular fractionation was used to observe the effect of SUMO-1 modification on the cytoplasmic/nuclear distribution of wild-type and polyQ-expanded ataxin-3.3.Co-immunoprecipitation was utilized to analyze the influence of SUMO-1 modifcation on the ubiquitination of wild-type and polyQ-expanded ataxin-3.4.Chase experiment was undertaken to investigate the effect of SUMO-1 modification on the protein degradation of wild-type and polyQ-expanded ataxin-3.5.Fluorescence technique,flow cytometry of PI/Annexin-V-FITC were used to study the effect of SUMO-1 modification on the formation of intranuclear inclusions,and apoptosis of wild-type and polyQ- expanded ataxin-3.Results:1.DNA sequencing confirmed that eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 were altered on the designed mutation loci,and the repeat number of CAG trinucleotide were 20 and 68 on wild-type and polyQ-expanded respectively,which the other loci matched with GenBank standard sequence(S75313).It suggested that eukaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frame shift after purpose gene order insertion.2.We identified eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3 was expressed in HEK 293T cell by Western-blot.Using GFP fluorescence technique,we found that ataxin-3-20Q and ataxin-3-20Q-K166R distributed scattered;on the contrast,ataxin-3-68Q and ataxin-3-68Q-K166R aggregated.3.We found SUMO-1 modification had no influence on the cytoplasmic/nuclear distribution of wild-type and polyQ- expanded ataxin-3 by subcellular fractionation.4.By co-immunoprecipitation,our study suggested that over-expression of SUMO-1 or SUMO-1 modification of missing,the polyubiquitination level between the wild-type and polyQ-expanded ataxin-3 protein was significantly different, including polyQ-expanded ataxin-3 protein polyubiquitination higher level.Over-expression of SUMO-1 or SUMO-1 modification of missing,the wild-type ataxin-3 protein polyubiquitination level had no difference,and the result was similar for polyQ-expanded ataxin-3 protein.5.We discovered by chase experiment that the bands weren't significantly different between ataxin3-20Q and ataxin3-20Q-K166R in 0h,1h,3h,7h,and 15h;but the bands of ataxin3-68Q were predominantly thicker than ataxin-3-68Q-K166R in 7h and 15h,which suggested that SUMO-1 modification resulted in stabilization of ataxin3-68Q.6.We analyzed the quantitation of intranuclear inclusion of the wild-type and polyQ-expanded ataxin-3 had significant different (P＜0.005),indicated that polyQ expansion could induce formation of the intranuclear inclusion;the means of wild-type ataxin-3 group or polyQ-expanded ataxin-3 group weren't significantly different between with/without SUMO-1 or excessive/native SUMO-1 modification.7.The result from flow cytometry of PI/Annexin-V/FITC showed the early apoptosis rate of ataxin-3-68Q group was higher than ataxin-3-68Q-K166R group(P＜0.005),which suggested that SUMO-1 modification had toxic effect;but the early apoptosis rate of ataxin-3-20Q group and ataxin-3-20Q-K166R group wasn't significantly different(P＞0.005). Conclusion:1.We construct pEGFP-N1 eukaryotic expression plasmids of the wild-type and polyQ-expanded ataxin-3 firstly,and obtain 14 HEK 293T cell strains.2.We confirm that SUMO-1 modification has no influence on the cytoplasmic/nuclear distribution of the wild-type and polyQ-expanded ataxin-3.3.We identify that K166 is not the ubiquitination locus of wild-type and polyQ-expanded ataxin-3.4.We discover that SUMO-1 modification resulted in stabilization of polyQ-expanded ataxin-3.5.We disclose that SUMO-1 modification of polyQ-expanded ataxin-3 had toxic effect,but didn't have influence on the nuclear inclusion formation.