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Purification of full-length recombinant human huntingtin proteins with allelic series of polyglutamine lengths

ABSTRACT: Summary Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the polyglutamine (polyQ) expansion in huntingtin (HTT) protein. The challenge of obtaining full-length HTT proteins with high purity limits the understanding of the HTT protein function. Here, we provide a protocol to generate and purify full-length recombinant human HTT proteins with various polyQ lengths, which is key to investigate the biochemical function of HTT proteins and the molecular mechanism underlying HD pathology. For complete details on the use and execution of this protocol, please refer to Jung et al. (2020). Graphical abstract Highlights • Expression and purification of full-length recombinant human huntingtin (HTT) proteins• Generation of HTT proteins with allelic series of polyglutamine length• Purified HTT proteins form complexes with HAP40 or HAP1. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the polyglutamine (polyQ) expansion in huntingtin (HTT) protein. The challenge of obtaining full-length HTT proteins with high purity limits the understanding of the HTT protein function. Here, we provide a protocol to generate and purify full-length recombinant human HTT proteins with various polyQ lengths, which is key to investigate the biochemical function of HTT proteins and the molecular mechanism underlying HD pathology.

SUBMITTER: Kim H

PROVIDER: S-EPMC8551504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice.

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Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features for Wild-Type and Pathological Polyglutamine Lengths.

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Project description:Repeat length disease thresholds vary among the 10 expanded polyglutamine (polyQ) repeat diseases, from about 20 to about 50 glutamine residues. The unique amino acid sequences flanking the polyQ segment are thought to contribute to these repeat length thresholds. The specific portions of the flanking sequences that modulate polyQ properties are not always clear, however. This ambiguity may be important in Huntington's disease (HD), for example, where in vitro studies of aggregation mechanisms have led to distinctly different mechanistic models. Most in vitro studies of the aggregation of the huntingtin (HTT) exon1 fragment implicated in the HD mechanism have been conducted on inexact molecules that are imprecise either on the N-terminus (recombinantly produced peptides) or on the C-terminus (chemically synthesized peptides). In this paper, we investigate the aggregation properties of chemically synthesized HTT exon1 peptides that are full-length and complete, containing both normal and expanded polyQ repeat lengths, and compare the results directly to previously investigated molecules containing truncated C-termini. The results on the full-length peptides are consistent with a two-step aggregation mechanism originally developed based on studies of the C-terminally truncated analogues. Thus, we observe relatively rapid formation of spherical oligomers containing from 100 to 600 HTT exon1 molecules and intermediate formation of short protofibril-like structures containing from 500 to 2600 molecules. In contrast to this relatively rapid assembly, mature HTT exon1 amyloid requires about one month to dissociate in vitro, which is similar to the time required for neuronal HTT exon1 aggregates to disappear in vivo after HTT production is discontinued.

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2018-09-03 | PXD010958 | Pride

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2017-03-22 | PXD005641 | Pride

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2017-05-26 | PXD006302 | Pride

Proteome analysis of huntingtin knock-in allelic series - Muscle

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