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HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

ABSTRACT: Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease and illuminate the structural consequences of HTT polyglutamine expansion.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Johannes Hevler

LAB HEAD: Albert J. R. Heck

PROVIDER: PXD028415 | Pride | 2022-09-09

REPOSITORIES: Pride

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HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Project description:Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease and illuminate the structural consequences of HTT polyglutamine expansion.

2022-02-21 | PXD028313 | Pride

PTMs of HTT protein - Post-translational modifications (PTMs), identified on endogenous huntingtin, cluster within proteolytic domains between HEAT repeats

Project description:Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, causative of Huntington’s disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, however none of these studies were designed to systematically characterize PTMs on the endogenous full-length Htt protein.We found that full-length endogenous Htt, immunoprecipitated from HD knock-in mouse and human post mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free mass spectrometry, we identified around 40 PTMs, of which half are novel. These findings will be instrumental in the further assembling the Htt PTM framework, and validate PTMs of Htt as promising therapeutic targets for HD.

2017-06-29 | PXD005753 | Pride

Huntingtin polyglutamine expansion modulates protein interaction dynamics in the striatum

Project description:Huntington’s disease is a monogenic disorder, with only one known causative gene, huntingtin (HTT). Expansion of a trinucleotide (CAG) repeat within the HTT gene results in a mutant protein containing polyglutamine (polyQ) stretches. While mutant HTT is the primary driver of cellular degeneration in the brain, the molecular and cellular mechanisms are incompletely understood. To further understand the role of polyQ in disease pathogenesis, we studied the dynamics of HTT protein-protein interactions (PPIs) in the striatum of mice with wild-type (Q20) and mutant (Q140) HTT at pre-symptomatic and manifest HD ages using label-free and isotope-labeled IP-MS approaches. Combining these approaches, we demonstrated that HTT PPI dynamics are distinct in early and later disease phases. Computational analysis pointed to early protein interaction changes involving synaptic transmission and vesicle fusion functions, while later interactions underlie changes in synapse morphogenesis and impact the actin cytoskeletal network.

2022-02-20 | PXD025510 | Pride

Identification of full length wild-type and mutant huntingtin interacting proteins by crosslinking immunoprecipitation in mice brain cortex

Project description:Huntington’s Disease is a neurodegenerative disorder caused by a CAG expansion in exon1 of the huntingtin (Htt) gene. The resulting polyglutamine expansion in the ubiquitously expressed mutant Htt (mHtt) protein leads to selective neurodegeneration. In this study we set out to identify interacting proteins of full length wild-type and mHtt protein in the mice cortex brain region in order to get a better understanding of the processes involved in HD pathology.

2021-06-21 | PXD024254 | Pride

Extensive changes in DNA methylation are associated with expression of mutant huntingtin [mRNA-seq]

Project description:The earliest stages of Huntington’s disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin protein (HTT). To explore the hypothesis DNA methylation may be altered in cells expressing mutated HTT, we use reduced-representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. Based on the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and at later stages, cognitive decline in Huntington’s patients. mRNA-seq in STHdhQ7/Q7 and STHdhQ111/Q111 cells

2013-01-24 | E-GEOD-43431 | biostudies-arrayexpress

Extensive changes in DNA methylation are associated with expression of mutant huntingtin [RRBS]

2013-01-24 | E-GEOD-43432 | biostudies-arrayexpress

Extensive changes in DNA methylation are associated with expression of mutant huntingtin [MeDIP-seq]

2013-01-24 | E-GEOD-43430 | biostudies-arrayexpress

Site-Specific Ubiquitination of Pathogenic Huntingtin Attenuates its Deleterious Effects

Project description:Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a CAG triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) stretch in the protein product which becomes aggregation-prone. The mutant Htt (mHtt) aggregates are associated with components of the Ubiquitin-Proteasome System (UPS), suggesting that mHtt is marked for proteasomal degradation and that for reasons still debated are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and its subsequent cellular response. We identified two lysine residues - 6 and 9 - in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size, but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Furthermore, proteomic analysis indicated that compared to cells expressing the protein that can be modified by ubiquitin, cells expressing the lysine-less protein did not activate protein synthetic pathways that enable the cell to cope with stress. Taken together, the findings suggest a novel role for ubiquitination - attenuation of the pathogenic effect of mHtt.

2020-06-30 | PXD019553 | Pride

Site-Specific Ubiquitination of Pathogenic Huntingtin Attenuates its Deleterious Effects- PART2

2020-06-30 | PXD019552 | Pride

Experimental replacement therapy counters decreased IGF-1 levels in Huntington’s disease

Project description:Huntington’s disease (HD) is a monogenetic neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in huntingtin (htt). Here we show that mutant htt reduces the transcription of insulin-like growth factor 1 (IGF-1) and leads to loss of IGF-1 in HD brains, HD mouse models and mutant htt-transgenic microglial cells. IGF-1 replacement therapy by transplantation of genetically engineered mouse neuronal precursor cells (mNPCs) in a mouse model of HD reverted the motor phenotype and countered striatal neuronal loss.

2018-10-01 | GSE19291 | GEO

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