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

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.

View Accepted Manuscript (DOE)

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

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

OSTI ID:: 1572550

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

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Proteogenomics connects somatic mutations to signalling in breast cancer Ping, Yan,; Francesca, Petralia,; Pei, Wang, The University of North Carolina at Chapel Hill University Libraries https://doi.org/10.17615/mepw-5x28	text	January 2016
Method to Introduce Stable, Expanded, Polyglutamine-Encoding CAG/CAA Trinucleotide Repeats into CAG Repeat-Containing Genes Michalik, Andrej; Kazantsev, Aleksey; Broeckhoven, Christine Van BioTechniques, Vol. 31, Issue 2 https://doi.org/10.2144/01312bm02	journal	August 2001
Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function Vijayvargia, Ravi; Epand, Raquel F.; Leitner, Alexander ETH Zurich https://doi.org/10.3929/ethz-b-000114979	text	January 2016
Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis Ratovitski, Tamara; Chighladze, Ekaterine; Arbez, Nicolas Cell Cycle, Vol. 11, Issue 10 https://doi.org/10.4161/cc.20423	journal	May 2012
Topological and energetic factors: what determines the structural details of the transition state ensemble and "on-route" intermediates for protein folding? An investigation for small globular proteins Clementi, Cecilia; Nymeyer, Hugh; Onuchic, Jose' Nelson arXiv https://doi.org/10.48550/arxiv.cond-mat/0003460	preprint	January 2000
Huntington disease: natural history, biomarkers and prospects for therapeutics Ross, Christopher A.; Aylward, Elizabeth H.; Wild, Edward J. Nature Publishing Group https://doi.org/10.5167/uzh-105328	text	January 2014
Huntingtin’s spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function Vijayvargia, Ravi; Epand, Raquel; Leitner, Alexander eLife, Vol. 5 https://doi.org/10.7554/eLife.11184	journal	March 2016

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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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