A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.
Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Jonasz J
JJ
Weber
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Matthias
M
Golla
Center for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany.|German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
Giambattista
G
Guaitoli
Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway.
Pimthanya
P
Wanichawan
German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.|Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research and Center of Neurology, University of Tübingen, Tübingen, Germany.
Stefanie N
SN
Hayer
German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
Stefan
S
Hauser
Paediatric Hematology and Oncology, University Children's Hospital Tübingen, Tübingen, Germany.
Ann-Christin
AC
Krahl
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Maike
M
Nagel
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.|Werner Reichardt Centre for Integrative Neuroscience, University of Tübingen, Tübingen, Germany.
Sebastian
S
Samer
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Department of (Neuro)Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.|Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlands.
Eleonora
E
Aronica
Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Oslo, Norway.
Cathrine R
CR
Carlson
German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.|Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research and Center of Neurology, University of Tübingen, Tübingen, Germany.
Ludger
L
Schöls
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Olaf
O
Riess
Center for Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany.|German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
Christian J
CJ
Gloeckner
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Huu P
HP
Nguyen
Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.|Centre for Rare Diseases, University of Tübingen, Tübingen, Germany.
Jeannette
J
Hübener-Schmid
D000067699:Ataxin-3 / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D003198:Computer Simulation; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism; D017827:Machado-Joseph Disease / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D014162:Transfection
D010447:Peptide Hydrolases; D000067699:Ataxin-3; D002154:Calpain
Machado-Joseph disease; ataxin-3; calpains; cleavage sites; spinocerebellar ataxia type 3