The Pathogenesis Of Parkin-mediated Non-classical Ubiquitination Of Mutant Ataxin-3 In SCA3

BackgroundSpinocerebellar ataxia type 3(SCA3),or Machado-Joseph disease,is the most common inherited spinocerebellar ataxia,caused by an expanded stretch of CAG repeats in the exon10 of ATXN3 gene.ATXN3 gene encodes ataxin-3 protein,which is a ubiquitously expressed deubiqutinating enzyme.Mutated ATXN3 results in the formation of an expanded polyQ tract in the protein,which will misfold and form ubiquitin-positive inclusion bodies in the neurons.The molecular mechanisms underlying the SCA3 disease remain elusive,and no preventive treatment is currently available.Ubiquitination is a common post-translational modification in which ubiquitin molecules bind to substrates through a series of enzymatic reactions,playing a critical role in maintaining protein quality control systems.Classical ubiquitination induces the degradation of substrates through proteasomes via the K48-linked ubiquitination.In addition,non-classical ubiquitination,such as K63-specific ubiquitination,induces autophagy signals and promotes the degradation of substrates through the autophagolysosomal pathway.The deubiquitinase activity of ataxin-3 and the ubiquitin-positive inclusion bodies formed by the mutation ataxin-3 highly suggest that abnormal ubiquitination may play a critical role in the pathogenesis of SC A3.PARK2,an associated gene with for autosomal recessive Parkinson’s disease,encodes parkin protein.Parkin is a RBR(RING-between-RING)E3 ubiquitin ligase that catalyzes various key substrates to form different ubiquitinated chains(such as K48-,K63-linked chains),and degrades the substrates through proteasome and/or lysosome.Thus,parkin is a crucial regulator in both ubiquitin-proteasome pathway and autophagy-lysosomal pathway.Previous studies showed parkin has widely involved in the pathogenesis of neurodegenerative diseases,such as Parkinson’s disease,Alzheimer’s disease,and Amyotrophic Lateral Sclerosis.The pathogenesis of SCA3 mediated by parkin still remains unclear.ObjectivesTo explore the mechanism of the polyubiquitination on expanded ataxin-3 mediated by parkin and its role on the degradation and aggregation of the mutant ataxin-3.Meanwhile,we will explore the the regulation of mutated ataxin-3 on the parkin expression.Methods1.Co-immunoprecipitation experiments were performed to explore the interaction between parkin and wild-type/mutant ataxin-3 and to expore the effect of the mutated PolyQ domain on its interaction strength with parkin in the SCA3 cellular models.The ubiquitination as well as types of the ubiquitin chains of wild-type/mutant ataxin-3 mediated by parkin were detected through via in vivo ubiquitination assays.2.The regulations of parkin on the stability as well as aggresome formation and clearance of mutant ataxin-3 were investigated in SCA3 cellular models;the roles of parkin protein in the cytotoxicity of mutant ataxin-3 protein were further analyzed.3.The protein stability of parkin regulated by mutant ataxin-3 was studied and the regulation of decreased parkin on the ubiquitination level,protein distribution and cytotoxicity of mutant ataxin-3 protein were analyzed.4.The protein level of parkin and ubiquitin as well as protein proteasome activity were detected in the brain of SCA3 transgenic mice.Results1.Both wild-type and mutant ataxin-3 protein interacted with parkin and the expansion of the ataxin-3 enhanced its interaction with the parkin.Both wild-type and mutant PolyQ domain interacted with parkin and the expansion of the polyQ domain directly enhanced its interaction with the parkin.Overexpression of parkin promoted ubiquitination of mutant ataxin-3,but not wild-type ataxin-3;Parkin preferentially ubiquitinated mutant ataxin-3 protein by K63-Ub chains.2.Parkin promoted the degradation of mutant ataxin-3 protein through autophagy lysosome system,reducing the level of mutant ataxin-3 protein,and alleviating the cytotoxicity induced by mutant protein.Under the condition of proteasome inhibition,parkin promoted aggresome formation and autophagic clearance of mutant ataxin-3.3.Mutation of ataxin-3 promoted the degradation of parkin through autophagolysosomal lysosomes system,reducing parkin level.Downregulation of parkin disturbed the ubiquitination of mutant ataxin-3,disturbing the formation and degradation of mutant protein aggregates,leading to an increased level of mutant ataxin-3 protein,and further aggravating the cytotoxicity induced by mutant ataxin-3.4.The level of parkin protein in the brain of SC A3 transgenic mice decreased,and the total ubiquitination level of K63 increased.Proteasome impairemet was detected in the brain of SCA3 transgenic mice.Conclusion1.Expanded ataxin-3 enhances its interaction with parkin;parkin ubiquitinates expanded ataxin-3 through K63-specific ubiquitin chain.2.Parkin could paly a a protective role by promoting aggresome formation and autophagic clearance of mutant ataxin-3 protein.3.Mutated ataxin-3 protein reduces the level of parkin protein by autophagy lysosomes pathway,disturbing the ubiquitination and clearance of mutant ataxin-3 mediated by parkin,which further aggravates the cytotoxicity induced by the mutant ataxin-3 protein.4.Parkin level decreases and the K63-linked ubiquitin level increases in the brain of SCA3 mice.