Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

Harding, Rachel J.; Loppnau, Peter; Ackloo, Suzanne; Lemak, Alexander; Hutchinson, Ashley; Hunt, Brittany; Holehouse, Alex S.; Ho, Jolene C.; Fan, Lixin; Toledo-Sherman, Leticia; Seitova, Alma; Arrowsmith, Cheryl H.

doi:10.1074/jbc.ra118.007204

Title: Design and characterization of mutant and wildtype huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

Journal Article · Wed Mar 06 00:00:00 EST 2019 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.ra118.007204· OSTI ID:1572550

^[1]; Loppnau, Peter ^[1]; Ackloo, Suzanne ^[1]; Lemak, Alexander ^[1]; Hutchinson, Ashley ^[1]; Hunt, Brittany ^[1];

^[2]; Ho, Jolene C. ^[1]; Fan, Lixin ^[3]; Toledo-Sherman, Leticia ^[4]; Seitova, Alma ^[1]; Arrowsmith, Cheryl H. ^[1]

Univ. of Toronto, ON (Canada)
Washington Univ., St. Louis, MO (United States)
National Institutes of Health (NIH), Frederick, MD (United States)
CHDI Foundation, Los Angeles, CA (United States)

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, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems in this work. 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.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE; National Institutes of Health (NIH); Natural Sciences and Engineering Research Council of Canada (NSERC); Huntington Society of Canada; CHDI Foundation; SGC Foundation; Ontario Genomics Institute; European Union (EU)

Grant/Contract Number:: HHSN26120080001E; RGPIN-2015-05939; OGI-055; 115766

OSTI ID:: 1572550

Journal Information:: Journal of Biological Chemistry, Vol. 294, Issue 17; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 11 works

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The pathobiology of perturbed mutant huntingtin protein–protein interactions in Huntington's disease Wanker, Erich E.; Ast, Anne; Schindler, Franziska Journal of Neurochemistry, Vol. 151, Issue 4 https://doi.org/10.1111/jnc.14853	journal	September 2019

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