Towards a Structural Understanding of the Fibrillization Pathway in Machado-Joseph's Disease: Trapping Early Oligomers of Non-expanded Ataxin-3

Abstract

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.

The Two-stage Pathway of Ataxin-3 Fibrillogenesis Involves a Polyglutamine-independent Step

“…Furthermore, morphological analysis revealed the formation of amyloid-like fibrils, which closely resemble those formed by the isolated Josephin domain (29). These data are consistent with previous studies of non-expanded ataxin-3, under physiological and denaturing conditions, which have also suggested that this protein has an intrinsic tendency to aggregate (32,33). The biological relevance of this in-built aggregation propensity, displayed by both non-expanded ataxin-3 and the Josephin domain (29), to the disease process is unclear.…”

“…Similar spheroids and oligomers have previously been shown to form from non-expanded ataxin-3 and poly(Q) peptides, with antibody analysis indicating that such pre-fibrillar aggregates may share both a common structure and toxic mechanism (33,43,54). Therefore, the neuronal toxicity observed in SCA3 is potentially caused by the ability of ataxin-3 to form these toxic structures.…”

“…Furthermore, numerous ataxin-3 variants with non-pathological length poly(Q) tracts have been shown to form amyloid-like aggregates under both near-physiological conditions (33) and denaturing conditions (19, 29 -31). Therefore, it is possible that the protein region responsible for the formation of ThT-positive aggregates involves the N-terminal Josephin domain, which is common to all of the proteins, although further work is needed to clarify this (29,33).…”

The RCK2 domain of the human BK Ca channel is a calcium sensor

“…Similar ligand-induced structural transitions have been reported for endoplasmic reticulum chaperones (42) and for a zincbinding protein (43). Typically, conformational transitions resulting in an increase in ␤ structure have been interpreted as a prelude to protein-protein interactions (42,44,45), and the ␣-to-␤ conformational transitions may occur at the interfaces between interacting domains of oligomeric structures (46). According to this view, the conversion of the protein to an increased ␤ structure would represent a transition favoring interaction between RCK domains.…”

Expansion of amino acid homo-sequences in proteins: Insights into the role of amino acid homo-polymers and of the protein context in aggregation

“…An important point, which is still controversial, is the mechanism that leads from expansion to pathology: whether homo-sequences are all that is needed to induce toxic aggregation and whether the rest of the protein sequence has any role in aggregate formation. Various independent reports have shown that some of the polyQ proteins tend to aggregate even in their non-expanded form and that protein context has a strong effect on the solubility and stability of polyQ stretches [8][9][10][11][12]. At least two of the polyQ proteins linked to pathologies have also been shown to contain other regions, distinct from the polyQ tract, that are able to aggregate and lead to amyloid formations similar to those observed in other degenerative diseases also when produced independently from the polyQ region or in their non-expanded form [9][10][11].…”