The Effect On The Subcellular Localization And Transrepression Activity Of Ataxin-3 Modified By SUMO-1

Background:The hereditary spinocerebellar ataxias(SCAs)are a heterogeneous group of neurodegenerative disorders.To date,at least 30 gene loci responsible for SCAs have been mapped,in which 17 pathogenic genes have been cloned.Among them,spinocerebellar ataxia type 3/ Machado-Joseph disease(SCA3/MJD)is the most common genotype. 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.Similar to ubiquitin,SUMO attachment to proteins which referred to as "sumoylation",enters a multi-step enzymatic pathway with pre-SUMO synthesis,hydrolytic activation,covalent conjugation and deconjugation.This reversible pathway provides a rapid and efficient way to modulate much prominent and basic function,such as subcellular localization,nuclear transport,transcriptional regulation, DNA repair,genomic integrality and signal transduction.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 this family of neurological 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. By online sumoylation analysis protocol,we found that ataxin-3 contains sumoylation motif(165VKGD168)at K166 and other potential lysine for SUMO-1 modification such as K8,K206.Objective:To identify the precise lysine of ataxin-3 for SUMO-1 modification, the effect on the subcellular localization and transrepression activity of ataxin-3 modified by SUMO-1.Methods:1.Overlap and long primer methods were undertaken to amplify MJD1 for site-specific mutagenesis.We constructed the eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3-K8R, ataxin-3-K166R,ataxin-3-K206R by using recombinant DNA technology.2.Immunofluorescence and Western-blot were used to detect the expression of eukaryotic expression plasmids.3.By Ni-NTA precipitation and Western-blot,we identified the precise lysine of ataxin-3 for SUMO-1 modification. 4.Immunofluorescence-laser cofocalization was undertaken to observe the effect the effect on the subcellular localization of ataxin-3 modified by SUMO-1.5.Mammalian two-hybrid system(Stratagene)and double luciferase report gene detection system(Promega)were used to analyze the transrepression activation of ataxin-3 and the effect on the transrepression activity of ataxin-3 modified by SUMO-1.Results:1.DNA sequencing confirmed that eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3-K8R,ataxin-3-K166R, ataxin-3-K206R 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 frameshift after purpose gene order insertion.2.We identified eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3-K8R,ataxin-3-K166R,ataxin-3-K206R were expressed in HEK293T cell by western-blot.Using immunofluorescence,we found that wild ataxin-3-K8R, ataxin-3-K166R,ataxin-3-K206R distributed scattered,on the contrast,polyQ-expanded ataxin-3-K8R,ataxin-3-K 166R, ataxin-3-K206R aggregated in the nucleus.3.By Ni-NTA precipitation and western-blot,we didn't observe any strap of SUMO-1 interaction when cotransfected ataxin-3-20Q-K166R,ataxin-3-68Q-K166R and SUMO-1,but found straps of SUMO-1 interaction when cotransfected wild-type and polyQ-expanded ataxin-3-K8R,ataxin-3-K206R.It suggested that K166 is the key amino acid of ataxin-3 for SUMO-1 modification, neither K8 nor K206.4.We found that ataxin-3-20Q,ataxin-3-20Q-K166R in HEK293T and PC 12 cell distributed scattered without difference between cytoplasm and nucleus,and in PC12 cell we observed the axon growth under NGF induction.On the contrast,ataxin-3-68Q,ataxin-3-68Q-K166R in HEK293T and PC12 cell expressed in cytoplasm and nucleus,and aggregated in the nucleus by Immunofluorescence-laser cofocalization.The results suggested that SUMO-1 modification didn't change the subcellular localization of ataxin-3-20Q and ataxin-3-68Q.5.The luciferase activity analysis showed the enzymatic activity of groups of pCMV-BD-ataxin-3-20Q and pCMV-BD-ataxin-3-68Q obviously decreased on contrast of pCMV-BD group(P＜0.05).The enzymatic activity of pCMV-BD-ataxin-3-20Q-K166 group obviously decreased on contrast of pCMV-BD-ataxin-3-20Q group(P＜0.05). The enzymatic activity of pCMV-BD-ataxin-3-68Q-K166 group decreased on contrast of pCMV-BD-ataxin-3-68Q group(P＞0.05).Conclusion:1.For the first time we identify that K166 is the key amino acid of wild-type and polyQ-expanded ataxin-3 for SUMO-1 modification, neither K8 nor K206.2.For the first time we found that SUMO-1 modification doesn't change the subcellular localization of wild-type and polyQ-expanded ataxin-3.3.We confirm that both the wild-type and polyQ-expanded ataxin-3 have the transrepression activation,especially polyQ-expanded ataxin-3.4.For the first time we disclose that SUMO-1 modification suppresses the transrepression activation of ataxin-3 firstly.