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1.
Protein Expr Purif ; 152: 40-45, 2018 12.
Article in English | MEDLINE | ID: mdl-30036587

ABSTRACT

An expansion of the polyglutamine (polyQ) tract within the deubiquitinase ataxin-3 protein is believed to play a role in a neurodegenerative disorder. Ataxin-3 contains a Josephin catalytic domain and a polyQ tract that renders it intrinsically prone to aggregate, and thus full-length protein is difficult to characterize structurally by high-resolution methods. We established a robust protocol for expression and purification of wild-type and expanded ataxin-3, presenting 19Q and 74Q, respectively. Both proteins are monodisperse as assessed by analytical size exclusion chromatography. Initial biophysical characterization was performed, with apparent transition melting temperature of expanded ataxin-3 lower than the wild-type counterpart. We further characterize the molecular envelope of wild-type and expanded polyQ tract in ataxin-3 using small angle X-ray scattering (SAXS). Characterization of protein-protein interactions between ataxin-3 and newly identified binding partners will benefit from our protocol.


Subject(s)
Ataxin-3/chemistry , Machado-Joseph Disease/genetics , Peptides/chemistry , Recombinant Proteins/chemistry , Repressor Proteins/chemistry , Ataxin-3/biosynthesis , Ataxin-3/genetics , Ataxin-3/isolation & purification , Chromatography, Gel/methods , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/pathology , Machado-Joseph Disease/metabolism , Machado-Joseph Disease/pathology , Models, Molecular , Peptides/metabolism , Protein Domains , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Scattering, Small Angle , X-Ray Diffraction
2.
Neurochem Res ; 42(5): 1430-1437, 2017 May.
Article in English | MEDLINE | ID: mdl-28236214

ABSTRACT

Neurodegenerative diseases are considered to be distinct clinical entities, although they share the formation of proteinaceous aggregates and several neuropathological mechanisms. Increasing evidence suggest a possible interaction between proteins that have been classically associated to distinct neurodegenerative diseases. Thus, common molecular and cellular pathways might explain similarities between disease phenotypes. Interestingly, the characteristic Parkinson's disease (PD) phenotype linked to bradykinesia is also a clinical presentation of other neurodegenerative diseases. An example is Machado-Joseph disease (MJD), with some patients presenting parkinsonism and a positive response to levodopa (L-DOPA). Protein aggregates positive for α-synuclein (α-Syn), a protein associated with PD, in the substantia nigra of MJD models made us hypothesize a putative additive biological effect induced by expression of α-Syn and ataxin-3 (Atx3), the protein affected in MJD. Hence, in this study we analysed the influence of these two proteins (α-Syn and wild-type or mutant Atx3) on modified redox signaling, a pathological process potentially linked to both diseases, and also the impact of exposure to iron and rotenone in SH-SY5Y neuroblastoma cells. Our results show that both α-Syn and mutant Atx3 overexpression per se increased oxidation of dichlorodihydrofluorescein (DCFH2), and co-expression of these proteins exhibited additive effect on intracellular oxidation, with no correlation with apoptotic features. Mutant Atx3 and α-Syn also potentiated altered redox status induced by iron and rotenone, a hint to how these proteins might influence neuronal dysfunction under pro-oxidant conditions. We further show that overexpression of wild-type Atx3 decreased intracellular DCFH2 oxidation, possibly exerting a neuroprotective role.


Subject(s)
Ataxin-3/biosynthesis , Oxidative Stress/physiology , Repressor Proteins/biosynthesis , alpha-Synuclein/biosynthesis , Ataxin-3/genetics , Cell Line, Tumor , Gene Expression , Humans , Oxidation-Reduction , Repressor Proteins/genetics , alpha-Synuclein/genetics
3.
Brain ; 138(Pt 12): 3537-54, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26490332

ABSTRACT

Machado-Joseph disease is a progressive neurodegenerative disorder associated with the polyQ-expanded ataxin-3 (encoded by ATXN3), for which no therapy is available. With the aim of clarifying the mechanism of neurodegeneration, we hypothesized that the abnormally long polyQ tract would interact aberrantly with ataxin-2 (encoded by ATXN2), another polyQ protein whose function has recently been linked to translational regulation. Using patient's samples and cellular and animal's models we found that in Machado-Joseph disease: (i) ataxin-2 levels are reduced; and (ii) its subcellular localization is changed towards the nucleus. Restoring ataxin-2 levels by lentiviral-mediated overexpression: (i) reduced mutant ataxin-3 levels; and (ii) rescued behaviour defects and neuropathology in a transgenic mouse model of Machado-Joseph disease. Conversely (i) mutating the ataxin-2 motif that enables binding to its natural interactor and translation activator poly(A)-binding protein; or (ii) overexpressing poly(A)-binding protein, had opposite effects, increasing mutant ataxin-3 translation and aggregation. This work suggests that in Machado-Joseph disease, mutant ataxin-3 drives an abnormal reduction of ataxin-2 levels, which overactivates poly(A)-binding protein, increases translation of mutant ataxin-3 and other proteins and aggravates Machado-Joseph disease. Re-establishment of ataxin-2 levels reduces mutant ataxin-3 and alleviates Machado-Joseph disease pathogenesis opening a new avenue for therapeutic intervention in this and potentially other polyQ disorders.


Subject(s)
Ataxin-2/genetics , Ataxin-2/metabolism , Ataxin-3/genetics , Down-Regulation , Machado-Joseph Disease/genetics , Machado-Joseph Disease/metabolism , Mutation , Protein Biosynthesis , Animals , Ataxin-3/biosynthesis , Humans , Lentivirus/genetics , Machado-Joseph Disease/therapy , Mice , Mice, Transgenic , Poly(A)-Binding Proteins/metabolism
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