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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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			Action	DRS
	20201023_OR16_UM7_Hevle001_SA_HTTQ23HAP40_CSMs_E1.xlsx	Xlsx
	20201023_OR16_UM7_Hevle001_SA_HTTQ23HAP40_CSMs_E2.xlsx	Xlsx
	20201023_OR16_UM7_Hevle001_SA_HTTQ23HAP40_CSMs_E3.xlsx	Xlsx
	20201023_OR16_UM7_Hevle001_SA_HTTQ23HAP40_CSMs_E4.xlsx	Xlsx
	20201023_OR16_UM7_Hevle001_SA_HTTQ23HAP40_PhoX_E1.raw	Raw

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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.

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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.

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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.

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

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