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

ABSTRACT: 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).

SUBMITTER: Kim H

PROVIDER: S-EPMC8551504 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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

Kim Hyeongju H Hyun Kyung-Gi KG Lloret Alejandro A Seong Ihn Sik IS Song Ji-Joon JJ

STAR protocols 20211016 4

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 under ...[more]

PMID: 34746859

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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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Quantification assays for total and polyglutamine-expanded huntingtin proteins.

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

Project description:Huntington's disease is caused by expansion of a polyglutamine (polyQ) domain within exon 1 of the huntingtin gene (Httex1). The prevailing hypothesis is that the monomeric Httex1 protein undergoes sharp conformational changes as the polyQ length exceeds a threshold of 36-37 residues. Here, we test this hypothesis by combining novel semi-synthesis strategies with state-of-the-art single-molecule Förster resonance energy transfer measurements on biologically relevant, monomeric Httex1 proteins of five different polyQ lengths. Our results, integrated with atomistic simulations, negate the hypothesis of a sharp, polyQ length-dependent change in the structure of monomeric Httex1. Instead, they support a continuous global compaction with increasing polyQ length that derives from increased prominence of the globular polyQ domain. Importantly, we show that monomeric Httex1 adopts tadpole-like architectures for polyQ lengths below and above the pathological threshold. Our results suggest that higher order homotypic and/or heterotypic interactions within distinct sub-populations of neurons, which are inevitable at finite cellular concentrations, are likely to be the main source of sharp polyQ length dependencies of HD.

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Development of mAb-based polyglutamine-dependent and polyglutamine length-independent huntingtin quantification assays with cross-site validation.

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Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex.

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Aggregation behavior of chemically synthesized, full-length huntingtin exon1.

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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Quantifying Huntingtin Protein in Human Cerebrospinal Fluid Using a Novel Polyglutamine Length-Independent Assay.

Project description:BackgroundThe use of biomarkers has become a major component of clinical trial design. In Huntington's disease (HD), quantifying the amount of huntingtin protein (HTT) in patient cerebrospinal fluid (CSF) has served as a pharmacodynamic readout for HTT-lowering therapeutic approaches and is a potential disease progression biomarker. To date, an ultrasensitive immunoassay to quantify mutant HTT protein (mHTT) has been used, but additional assays are needed to measure other forms of HTT protein.ObjectiveWe aimed to develop an ultrasensitive immunoassay to quantify HTT protein in a polyglutamine length-independent manner (mHTT and non-expanded wild type HTT combined) in control and HD participant CSF samples.MethodsAn ultrasensitive, bead-based, single molecule counting (SMC) immunoassay platform was used for the detection of HTT protein in human CSF samples.ResultsA novel ultrasensitive SMC immunoassay was developed to quantify HTT protein in a polyglutamine length-independent manner and shown to measure HTT in both control and HD participant CSF samples. We validate the selectivity and specificity of the readout using biochemical and molecular biology tools, and we undertook a preliminary analytical qualification of this assay to enable its clinical use. We also used this novel assay, along with the previously described mHTT assay, to analyze CSF from control and HD participants. The results of this preliminary set suggests that correlation is present between mHTT and the polyglutamine length-independent HTT levels in human CSF.ConclusionWe have developed a novel ultrasensitive immunoassay that is able to quantify HTT protein in a polyglutamine length-independent manner in control and HD participant CSF.

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Proteome analysis of huntingtin knock-in allelic series - Heart

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2018-09-03 | PXD010958 | Pride

Proteome analysis of huntingtin knock-in allelic series - Cortex

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