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G3BP1-dependent suppression of polyglutamine aggregation in Huntington’s models and its failure upon stress granule formation

ABSTRACT: To define the interactome of huntingtin protein (HTT) in human neurons, we assessed interactors of HTT with coimmunoprecipitation experiments with huntingtin antibody or polyqlutamine antibody (it only recognizes mutant huntingtin protein with abnormal polyqlutamine expansion) in striatal neurons differentiated from control and patient-derived iPSC lines (HD-iPSCs). GFP antibody used as a negative control.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Huntington Disease

SUBMITTER: Prerana Wagle

LAB HEAD: David Vilchez

PROVIDER: PXD037618 | Pride | 2022-12-02

REPOSITORIES: pride

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			Action	DRS
	20170317_Q1_UC_ColID_319_ProjID_488_Vilchez_Sample_01.raw	Raw
	20170317_Q1_UC_ColID_319_ProjID_488_Vilchez_Sample_03.raw	Raw
	20170317_Q1_UC_ColID_319_ProjID_488_Vilchez_Sample_04.raw	Raw
	20170317_Q1_UC_ColID_319_ProjID_488_Vilchez_Sample_06.raw	Raw
	20170317_Q1_UC_ColID_319_ProjID_488_Vilchez_Sample_07.raw	Raw

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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

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.

2022-09-09 | PXD028415 | Pride

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

2022-02-21 | PXD028313 | Pride

PolyQ-expansion does not alter the huntingtin-HAP40 complex

Project description:The amplification of a CAG repeat in the gene coding for huntingtin (HTT) leads to Huntington’s disease (HD). At the protein level, this translates into the expansion of a poly-glutamine (polyQ) stretch in the HTT N-terminus, which renders HTT aggregation-prone by unknown mechanisms. Here we investigate the effects of polyQ expansion on the HTT-HAP40 complex, where HTT structure is substantially stabilized. Surprisingly, our biophysical, cryo-EM and crosslinking mass spectrometry experiments reveal no major changes between 17QHTT-HAP40 (wild type), 46QHTT-HAP40 (typical polyQ length in HD patients) and 128QHTT-HAP40 (extreme polyQ length). Thus, the proposed destabilizing effect of HTT polyQ expansion may not suffice to alter the HTT-HAP40 complex, suggesting that polyQ expansion does not have a major global effect on HTT structure.

2021-03-19 | PXD018451 | 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

The ubiquitin ligase UBR5 suppresses proteostasis collapse in immortal pluripotent stem cells and Huntington’s disease models

Project description:Pluripotent stem cells undergo unlimited self-renewal while maintaining their potential to differentiate into post-mitotic cells with an intact proteome, a capacity that demands a highly induced proteostasis network. As such, induced pluripotent stem cells (iPSCs) suppress the aggregation of polyQ-expanded huntingtin (HTT), the mutant protein underlying Huntington’s disease (HD). Here we show that proteasome activity determines HTT levels, preventing the accumulation of polyQ-expanded aggregates in iPSCs from HD patients (HD-iPSCs). iPSCs exhibit intrinsic high levels of UBR5, an E3 ubiquitin ligase that we find required for proteasomal degradation of both normal and mutant HTT. When UBR5 fails to monitor HTT proteostasis, the concomitant up-regulation of HTT expression particularly results in polyQ-expanded aggregation in HD-iPSCs. Moreover, UBR5 knockdown hastens protein aggregation and neurotoxicity in polyQ-expanded invertebrate models. Notably, ectopic expression of UBR5 is sufficient to induce polyubiquitination and degradation of mutant HTT, reducing polyQ-expanded aggregates in HD cell models. However, UBR5 is dispensable for the proteostasis of other aggregation-prone proteins linked with Machado-Joseph disease or amyotrophic lateral sclerosis in iPSCs. Besides its role in HTT regulation, we find that intrinsic high levels of UBR5 also determine the global proteostatic ability of iPSCs preventing aggresome formation, suggesting a role in the control of misfolded proteins ensued from normal metabolism. Thus, our findings indicate UBR5 as a central component of super-vigilant proteostasis of iPSCs with the potential to correct proteostatic deficiencies in HD.

2018-06-14 | PXD009803 | Pride

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

Mutant Huntingtin promotes neuronal death through cell autonomous microglial activation via myeloid lineage- determining factors

Project description:Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by an extended polyglutamine repeat in the N-terminus of the huntingtin (Htt) protein. Reactive microglia and elevated cytokine levels are observed in the brains of HD patients, but the extent to which neuroinflammation results from extrinsic or cell-autonomous mechanisms is unknown. Furthermore, the impact of microglia activation on the pathogenesis of HD remains to be established. Using genome-wide approaches, we show that expression of mutant Htt in microglia promotes cell-autonomous pro-inflammatory transcriptional activation within microglia by increasing the expression and transcriptional activities of the myeloid lineage-determining factors PU.1 and C/EBPs. Elevated levels of PU.1 and its target genes are observed in the brains of mouse models and HD individuals. Moreover, mutant Htt expressing microglia exhibit an increased capacity to induce neuronal death ex vivo and in vivo in the presence of sterile inflammation. These findings suggest that expression of mutant Htt in microglia may contribute to neuronal pathology in Huntingtin disease. RNA-Seq and ChIP-Seq for PU.1, C/EBP, and H3K4me2 in BV2 cells and RNA-Seq in primary microglia and macrophages

2014-03-02 | E-GEOD-54443 | biostudies-arrayexpress

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