Design and characterization of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems.

Design and characterization of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems.

Publication date: Mar 06, 2019

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 posttranslational 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 (SAXS), 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.

Harding, R.J., Loppnau, P., Ackloo, S., Lemak, A., Hutchinson, A., Hunt, B., Holehouse, A.S., Ho, J.C., Fan, L., Toledo-Sherman, L., Seitova, A., and Arrowsmith, C.H. Design and characterization of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems. 06384. 2019 J Biol Chem.

Concepts Keywords
Biochemical Small-angle X-ray scattering
Biomolecules PolyQ
Eukaryotic HD
Expression System Neurodegeneration
Glutamine HTT
Huntingtin Polyglutamine tract
Huntington Huntingtin
KDa Trinucleotide repeat disorder
Lengths Huntington’s disease
Milligram Branches of biology
Mutant Eukaryotic expression systems
PolyQ
Posttranslational Modifications
Protein
Recombinant
SAXS
Scattering
Wild Type
X Ray

Semantics

Type Source Name
disease DOID Huntington disease
disease MESH Huntington disease
gene UNIPROT RAB35
gene UNIPROT SH3YL1
gene UNIPROT AOPEP
gene UNIPROT SET
disease MESH multiple
gene UNIPROT SLC6A4
gene UNIPROT LARGE1
drug DRUGBANK L-Glutamine
gene UNIPROT HTT

Original Article

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