A Major Role for Side-Chain Polyglutamine Hydrogen Bonding in Irreversible Ataxin-3 Aggregation

Abstract

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm−1 in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.

Molecular Interaction between the Chaperone Hsc70 and the N-terminal Flank of Huntingtin Exon 1 Modulates Aggregation

“…Previously published structural analyses of polyQ fibrils performed mostly on synthetic polyQ showed no similar structural transition (49 -51). However, one group reported differences in the FTIR spectra of fibrils from recombinant ataxin 3 containing 24 or 55 glutamines (52). The apparent discrepancy between results obtained using synthetic polyQ peptides and recombinant polyQ proteins highlights the importance of the natural context of polyQ stretches.…”

Fourier Transform Infrared Spectroscopy of Intrinsically Disordered Proteins: Measurement Procedures and Data Analyses

“…Peaks due to amino acid side chain can be observed in the protein mid-infrared absorption spectrum within a wide range. Interestingly, the analyses of these bands can give useful information on the amino acid side chain conformation, such as exposure and H-bonding (41). A review of amino acid side chain absorptions in the mid-infrared can be found in ref.…”

Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome

“…These AT3 variants were expressed in E. coli strain BL21-CodonPlus (DE3)-RIL (E. coli B F− ompT hsdS (rB− mB−) dcm+ Tetr gal (DE3) endA Hte [argU ileY leuW Cam r ] (Stratagene, La Jolla, CA, USA) as GST-fusion proteins containing a PreScission Protease recognition site. The wild type AT3Q24 was cloned in a pQE30 vector (Qiagen Hamburg GmbH, Hamburg, Germany) and expressed as a His-tagged protein as previously described (Natalello et al, 2011). The variants AT3Q6 and AT3Q24-3UIM were obtained by PCR reactions using 5 -phosphorylated oligos and the construct pQE30/AT3Q24 as template.…”

“…Protein content was determined using Coomassie brilliant blue G-250 (Thermo Fisher Scientific, Rockford, IL, USA) and BSA as a standard protein. To ensure that the protein variants under investigation were properly folded, they were routinely subjected to FT-IR analysis to assess secondary structure content (Natalello et al, 2011).…”