Abstract
The gene mutated in individuals with Huntington's disease (HD) encodes the 348-kDa huntingtin (HTT) protein. Pathogenic HD CAG-expansion mutations create a polyglutamine (polyQ) tract at the N terminus of HTT that expands above a critical threshold of ∼35 glutamine residues. The effect of these HD mutations on HTT is not well understood, in part because it is difficult to carry out biochemical, biophysical, and structural studies of this large protein. To facilitate such studies, here we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprised both N- and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, and juvenile HD patients, as well as the more extreme polyQ expansions used in some HD tissue and animal models. Our expression system yielded milligram quantities of pure recombinant HTT protein, including many of the previously mapped post-translational modifications. We characterized both apo and HTT-HTT-associated protein 40 (HAP40) complex samples produced with this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful HTT complexes. We further demonstrate that these resources can produce sufficient material for protein-intensive experiments, such as small-angle X-ray scattering, providing biochemical insight into full-length HTT protein structure. The work outlined and the tools generated here lay a foundation for further biochemical and structural work on the HTT protein and for studying its functional interactions with other biomolecules.
Highlights
The gene mutated in individuals with Huntington’s disease (HD) encodes the 348-kDa huntingtin (HTT) protein
A Peptide–spectrum matches (PSMs) are reported as the number of peptide spectrum matches for the most abundant peptide containing the modification described with the total number of peptide spectra for all peptides containing this modification motif in parentheses
Some of these previously unreported modifications may be artifacts of the Sf9 expression system, they appear to have a minimal effect on global huntingtin function, as seen by the ability of this sample to form a complex with HTT-associated protein 40 (HAP40)
Summary
We identified HTT clones with a variety of polyQ lengths with both N- and C-terminal FLAG tags (Fig. 1B) These entry vectors serve as valuable reagents to allow future generation of even more polyQ length HTT constructs. Samples were analyzed by Western blotting using anti-HTT antibodies that revealed a discrete band of the expected molecular weight for each sample (Fig. S1) To assess whether these protein samples were folded, the C-terminal FLAG-tagged HTT samples of different polyQ lengths, purified from Sf9 cells, were analyzed by DSLS over a temperature gradient from 25 to 85 °C to assess thermal stability and propensity to aggregate under increasing temperatures. All data are available via PRIDE (accession PXD010865) with summaries in Zenodo
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