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.