A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3

Abstract

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.

Physiological and pathophysiological characteristics of ataxin-3 isoforms

“…This is of special interest, as it was previously reported that cleavage of mutant ataxin-3 leads to a nuclear translocation of the polyQcontaining fragment (4,9). At the same time, these fragments are considered to be more toxic and show an increased propensity to form aggregates compared with the full-length protein (2,24,26,27,47,89,90). Ataxin-3 is known to be a deubiquitinating enzyme (7,8), and that cellular turnover of ataxin-3, as well as its steady-state level, is regulated by its catalytic activity (13).…”

“…Subcellular localization of ataxin-3 was analyzed by a separation of homogenates into whole-cell, nuclear, and cytoplasmic fractions using the REAP fractionation protocol (105) with minor modifications (27).…”

“…One hallmark of MJD is the formation of macromolecular aggregates containing the pathological expanded ataxin-3 (4,24). Further, it is known that ataxin-3 is cleaved by calpains and caspases (25)(26)(27). Cleavage is required for aggregate formation in vitro and in vivo and may be part of the pathogenesis according to the toxic fragment hypothesis (28).…”

Impaired proteostasis in rare neurological diseases

“…By the use of hiPSC and functional neurons derived from SCA3 patients, it was demonstrated the key role of Ca 2+ -dependent calpain proteases in the proteolysis of CAG-expanded ataxin-3 [207]. In line with this, using fibroblast and iPSCs, it has recently been shown that calpain primarily cleaves ATXN3 in two specific sites, and that resulting fragments have distinct aggregation tendency [208]. Nevertheless, Hansen and co-workers could not replicate ATXN3 aggregate formation using their patient iPSC-derived neurons [209].…”

Roles of Post-translational Modifications in Spinocerebellar Ataxias

“… Schmidt et al (1998) discovered that NIs in neuronal SCA3 patient cells are created by fragments of pathogenic proteins, implying that ATXN3 undergoes proteolytic cleavage prior to its transport into the nucleus. ATXN3, a protein with a molecular weight of approximately 42 kDa, is proteolytically modified by caspases and calpains ( Berke et al, 2004 ; Weber et al, 2017 ). D241, D244, and/or D248 are the three most relevant caspase cleavage sites in ATXN3 ( Evers et al, 2014 ), and the two major calpain cleavage sites occur at amino acids D208 and S256 ( Weber et al, 2017 ) ( Figure 1B ).…”