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

Abstract

The gene mutated in Huntington's disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying 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. Our set of HTT expression clones comprises both N and C-terminally FLAGtagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural A toolkit of HTT protein resources 2 work on the HTT protein and its functional interactions with other biomolecules.

Translation in amino-acid-poor environments is limited by tRNAGln charging

“…PolyQ −1 -GFP reporter construct was built in a pCDH-CMV-MSC-EF1α-puro constitutive lentiviral vector (CD510B-1, System Biosciences). For the construction of PolyQ-GFP and PolyQ −1 -GFP reporter, the first exon of the human HTT allele (polyQ length = 36) was PCR-amplified from pBacMam2-DiEx-LIC-C-flag_huntingtin_full-length_Q36 construct (Addgene #111745, a gift from Dr. Cheryl Arrowsmith [ Harding et al, 2019 ]). Primers for the PCR amplification were designed to contain a 20 bp overlap with the corresponding vector backbone (forward primer) and with the ATG-less GFP ORF preceded by a flexible linker (reverse primer).…”

“…Arrowsmith (Harding et al, 2019)). Primers for the PCR amplification were designed to contain a 20-bp overlap with the corresponding vector backbone (forward primer) and with the ATGless GFP ORF preceded by a flexible linker (reverse primer).…”

The pathobiology of perturbed mutant huntingtin protein–protein interactions in Huntington's disease

“…HTT is a large protein of ~ 350 kDa that plays critical functional roles in gene expression regulation (Valor ), vesicle transport (Harjes and Wanker ) and autophagy (Ochaba et al ). It is modified post‐translationally at multiple sites (Cariulo et al ; Harding et al ) and gets cleaved by various proteases (Sanchez Mejia and Friedlander ), leading to the release of N‐terminal fragments with an expanded polyQ sequence, which have a high propensity to misfold and self‐assemble into fibrillar aggregates (Scherzinger et al ; Trepte et al ). As a consequence, intranuclear inclusions are formed in neurons.…”