Proteasomal Degradation Of Ataxin-3, A SCA3 Disease Protein

Spinocerebellar ataxia(SCA3),also called Machado-Joseph disease(MJD),is an autosomal dominant neurodegenerative disorder.It is the most common subtype in spinocerebellar ataxia.which is characterized clinically by cerebellar ataxia,pyramidal and extrapyramidal signs,progressive extraocular muscle paralysis and facial tic.It's reported that the mean age at onset is 37.4+14.1 years in the whole world,and in China the mean age is about 33.9+9.5 years.It is caused by an expansion of the polyglutamine tract near the C-terminus of the MJD-1 gene product,ataxin-3.Ataxin-3 is ubiquitously expressed in the body.The hallmark of MJD,as well as other polyglutamine diseases,is the formation of high ubiquitinated nuclear inclusions in affected brain,resulting from an abnormal accumulation of misfolded polyglutamine protein,along with molecular chaperones,transcription factors or co-activators and the components of proteasome.The presence of above proteins to inclusions formed by expanded ataxin-3 suggests that a functional alteration or impairment of those proteins may occur,thus contributing to MJD pathogenesis.The ubiquitin proteasome pathway(UPP) is a proteolysis route for most non-lysosomal degradation proteins,including both short-lived regulatory proteins and constitutively stable cellular proteins with long half-lives,as well as misfolded and damaged polypeptides.Usually,the proteins are polyubiquitinated by a multistep process,involving at least three types of enzyme called a ubiquitin-activating enzyme (E1),a ubiquitin-conjugating enzyme(E2),and a ubiquitin-protein ligase(E3) before they are degraded by the proteasome.The 19S subcomplex is composed of several non- ATPase subunits and six ATPase subunits to function in selective degradation of proteins and regulation of the activity of the 20S proteasome.The functional specificity of different proteasomal ATPase subunits remains unclear,but mutations in individual ATPase subunits cause different phenotypes and reduce the degradation of some substrates,while not affecting others.Within the six ATPase subunits,p45 has been shown to interact with Sp1 and vitamin D receptor(VDR) and to enhance the degradation of Sp1 and VDR specifically by the proteasome both in vitro and in vivo.Ataxin-3 contains two or three ubiquitin-interacting motifs(UIMs) based on its splice variant,functioning in modulating the de-ubiqutination of its substrates through its Joseph domain,a ubiquitin protease(UBP).Ataxin-3 also interacts with proteins functionally associated with UPP,such as HHR23 proteins and valosin-containing protein(VCP/p97),suggesting that there are tight links between ataxin-3 and the proteasome.Studies have shown that ataxin-3 is degraded by the proteasome, however,the precise mechanism of the degradation of ataxin-3 is still unknown.Here we show a direct link between ataxin-3 and the proteasome.Using in vitro and in cells studies,p45,an ATPase subunit of the 26S proteasome,interacts with normal ataxin-3 as well as expanded ataxin-3 directly.In reconstituted degradation assay,p45 stimulates the degradation of ataxin-3 by the proteasome.The regulation can be blocked by MG-132.In N2a cells or 293 cells,overexpression of p45 strikingly enhances the clearance of both normal and expanded ataxin-3,but not alpha synuclein or SOD1,implying a functional specificity of p45 in this proteolytic process. In contrast,an ATPase mutant of p45,which is still able to bind ataxin-3,loses the ability to induce the degradation of ataxin-3.Furthermore,we confirmed that the N-terminus of ataxin-3 interacts with p45 and demonstrated that the degradation of ataxin-3 driven by p45 depends on the presence of its UIMs,but the UIMs are not sufficient for this regulation.As p45 is an ATPase of the 19S proteasome,we wonder whether other ATPase of the 19S proteasome also promotes the degradation of ataxin-3.The data suggest that p45 specifically drives the process.Thus our findings suggest that the ATPase subunit of the 19S proteasome p45 can specifically regulated the degradation of polyglutamine protein ataxin-3,and this effect is through the proteasome pathway.The UIMs of ataxin-3 is not sufficient but important.The binding sites are the N-termins.The findings here will be of help to expand our knowledge of ataxin-3 functions,as well as its possible role in SCA3.