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Integrated Tandem Affinity Protein Purification Using the Polyhistidine plus Extra 4 Amino Acids (HiP4) Tag System２

ABSTRACT: Interactome analysis of TARDBP (TDP-43) using a novel tag system (HiP4-tag system).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Akihide Ryo

PROVIDER: PXD038445 | JPOST Repository | Tue Feb 28 00:00:00 GMT 2023

REPOSITORIES: jPOST

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			Action	DRS
	20221118QE_TDP43_HisOnly_H2O2_1.raw	Raw
	20221118QE_TDP43_HisOnly_H2O2_2.raw	Raw
	20221118QE_TDP43_HisOnly_H2O2_3.raw	Raw
	20221118QE_TDP43_HisOnly_cont_1.raw	Raw
	20221118QE_TDP43_HisOnly_cont_2.raw	Raw

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Publications

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system.

Ino Yoko Y Yamaoka Yutaro Y Tanaka Kiho K Miyakawa Kei K Nishi Mayuko M Hatayama Yasuyoshi Y Kimura Hirokazu H Kimura Yayoi Y Ryo Akihide A

Proteomics 20230303 11

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed "HiP4" (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this s ...[more]

PMID: 36807525

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Integrated Tandem Affinity Protein Purification Using the Polyhistidine plus Extra 4 Amino Acids (HiP4) Tag System

Project description:Interactome analysis of HBx using a novel tag system (HiP4-tag system).

2023-02-28 | PXD036256 | JPOST Repository

TDP-43 Interactor and Motor Neuron Tagged Ribosome Affinity Purification in a Drosophila Model of ALS

Project description:We report the enrichment of mRNAs with motor neuron specific TDP-43 and tagged ribosomes in control as well as multiple models of TDP-43 induced neurodegeneration. Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. The goal of this study is to understand the translational consequences of TDP-43 pathology. Using the GAL4-UAS system, we expressed TDP-43WT and TDP-43G298S in the motor neurons of our drosophila to induce ALS-like neurodegeneration. TDP-43 was immunoprecipitated from the larvae at the 3rd instar stage; TDP-43 associated mRNAs, the whole larvae input, and ventral nerve cords of the same genotype were then sequenced. To identify translational changes we conducted TRAP (translating ribosome affinity purifications) in 3rd instar larvae by immunoprecipitating the motor neuron specific tagged ribosomal subunit RpL10-GFP in both ALS models as well as a RpL10-GFP control. The RpL10-GFP associated mRNAs, the whole larval input, and ventral nerve cords of the same genotype. From this data, we identified both compensatory alterations to translation induced by TDP-43 pathology and direct targets of TDP-43 mediated translational inhibition.

2021-03-31 | GSE156222 | GEO

Decrease of Parkin and CD36 expression and body weight correlate with an increase of non-esterified fatty acids in a mouse model for TDP-43 pathology

Project description:TDP-43-coding mutations are found among ALS patients. In oder to express human mutant TDP-43 protein in the mouse under the control of the endogenous Tardp promoter, we have generate a mouse line wit 300% elevated TDP-43 level.

2014-05-21 | GSE39585 | GEO

Decrease of Parkin and CD36 expression and body weight correlate with an increase of non-esterified fatty acids in a mouse model for TDP-43 pathology

2014-05-21 | E-GEOD-39585 | biostudies-arrayexpress

Identifying modifiers of seed-induced TDP-43 loss of function

Project description:Both gain of toxicity and loss of normal function of the RNA-binding protein TDP-43 contribute to neurodegeneration in ALS and FTD, but their mechanistic connection remains unclear. Increasing evidence suggests that TDP-43 aggregates act as self-templating seeds, propagating pathology through the central nervous system via a prion-like cascade. We developed a robust TDP-43 seeding platform for quantitative, high-throughput assessment of TDP-43 aggregate uptake, cell-to-cell spreading and direct quantification of TDP-43 nuclear function within living cells, while they progress towards pathology. We show that both patient-derived and recombinant TDP-43 pathological aggregates are abundantly internalized in human neuron-like cells, efficiently recruit endogenous TDP-43 and form cytoplasmic inclusions reminiscent of ALS/FTD pathology. These neoaggregates progressively drive the nuclear egress of TDP-43 leading to its loss of function. The scope of this project is to identify the signatures involved in the progressive egress of TDP-43 from the nucleus to the cytoplasm.

2025-02-20 | GSE285224 | GEO

TDP-43 represses a cryptic exon in Schwann cells to ensure rapid saltatory conduction

Project description:A pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia is the aggregation of TDP-43. While extensive exploration into the function of TDP-43 has focused entirely in the central nervous system (CNS), the implications in physiology and pathology of the peripheral nervous system (PNS) have long been overlooked. Herein, we demonstrate that deletion of TDP-43 in Schwann cells results in a 50% reduction in peripheral nerve conduction velocity, without any obvious alterations to peripheral compact myelin. Compromised insulatory function of myelin was due to the complete loss of paranodal axoglial junctions flanking the nodes of Ranvier. By contrast, the paranodal junctions in the CNS oligodendrocytes are unaltered upon deletion of TDP-43. Mechanistically, TDP-43 binds directly to Nfasc mRNA, which encodes neurofascin, a key cell adhesion molecule essential for establishing paranodal junctions. TDP-43 binding sites overlap with a cryptic exon of Nfasc such that loss of TDP-43 leads to the retention of this cryptic exon with a premature stop codon, a prerequisite for nonsense-mediated decay. Thus, TDP-43 is required for the proper assembly of nodes of Ranvier that is essential for saltatory conduction by suppressing the cryptic exon in the Nfasc mRNA of the Schwann cells. Our findings indicate a direct involvement of TDP-43 in normal PNS function and suggest that PNS-autonomous dysfunction in saltatory conduction may contribute to TDP-43 proteinopathies.

2021-09-01 | GSE157714 | GEO

Location and function of TDP-43 in platelets: Alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols

Project description:The TAR DNA Binding Protein (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Fractionation studies revealed that >95% of plasma TDP-43 protein [RL1] was located within the platelet cytosol, together with numerous RNAs. Platelet-derived TDP-43 exhibits TDP-43 proteoforms detected in neurodegenerative diseases, TARDBP RNA splice variants and TDP-43 RNA targets found in the central nervous system (CNS). We propose that TDP-43 serves similar functional roles in platelets and synapses, suggesting that the study of platelet TDP-43 might provide a window into TDP-43 proteinopathies within the CNS. The restricted compartmentalization of plasma TDP-43 in platelets provides a highly concentrated substrate for further biochemical analyses. Moreover, our results suggest that current plasma biobanking protocols are subject to considerable heterogeneity in platelet recovery and measurements of TDP-43 in plasma.

2024-09-25 | GSE245303 | GEO

Cell environment shapes TDP-43 function: implications in neuronal and muscle disease

Project description:TDP-43 aggregation and redistribution have been recognised as a hallmark of amyotrophic lateral sclerosis, frontotemporal dementia and other neurological disorders. While TDP-43 has been studied extensively in neuronal tissues, TDP-43 inclusions have also been described in the muscle of inclusion body myositis patients, highlighting the need to understand the role of TDP-43 beyond the central nervous system. Using RNA-seq we performed the first direct comparison of TDP-43-mediated transcription and alternative splicing in muscle (C2C12) and neuronal (NSC34) mouse cells. Our results clearly show that TDP-43 displays a tissue-characteristic behaviour targeting unique transcripts in each cell type. This is not due to variable transcript abundance but rather due to cell-specific expression of RNA-binding proteins, which influences TDP-43 performance. Among splicing events commonly dysregulated in both cell lines, we identified some that are TDP-43-dependent also in human cells and show that inclusion levels of these alternative sequences appear to be altered in affected tissues of FTLD and IBM patients. We therefore propose that TDP-43 dysfunction, reflected in aberrant splicing, contributes to disease development but it does so in a tissue-specific manner.

2022-04-27 | GSE171714 | GEO

Endothelial TDP-43 controls sprouting angiogenesis and vascular barrier integrity, and its deletion triggers neuroinflammation

Project description:TDP-43 is a DNA/RNA-binding protein that regulates gene expression and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its role in endothelial cells (ECs), angiogenesis and vascular function. Using inducible EC-specific TDP-43 knockout mice, we show that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity and blood vessel stability. Postnatal EC-specific deletion of TDP-43 leads to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupts the blood-brain barrier and triggers vascular degeneration. These vascular defects are associated with an inflammatory response in the central-nervous system with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupts fibronectin matrix around sprouting vessels and reduces -catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.

2024-02-01 | GSE253868 | GEO

Parallel high-throughput RNA-sequencing suggests little overlap of differential gene expression between knockout of TDP-43 and its over-expression in central nervous system in Drosophila

Project description:The human Tar-DNA-binding protein TDP-43 is closely associated with ALS and other neurodegenerative disorders. TDP-43 contains two highly conserved RNA-binding motifs and possesses a variety of documented roles in RNA metabolism, including pre-RNA splicing and repression of transcription. We sought to measure the effect that knockout and over-expression of the fly orthologue of this protein, Tar-DNA-binding protein homolog (TBPH), has on the transcriptome of the central nervous system (CNS) of Drosophila melanogaster. To this end, we used massively parallel sequencing methods (RNA-seq) to transcriptionally profile the CNS in loss-of-function mutants and gain-of-function over-expression genotypes. We found that loss of TBPH resulted in widespread gene activation, much of which could be reversed by rescue of TBPH expression, suggesting that repression is one of the major roles of TBPH. Conversely, we found that over-expression of TBPH resulted largely in decreased gene expression. However, there was little overlap in the genes which were affected in these two genotypes, suggesting that the bulk of genes affected by TBPH loss-of-function and over-expression are different. We provide a comprehensive look at enriched gene ontologies in both cases, suggesting that TDP-43 plays a role in regulating basic processes in neurons. We also describe a number of genes whose splicing is likely to be altered in the absence of TDP-43. In this study we compare the effects of knockout of the TDP-43 ortholog (TBPH) in the fly nervous system with the effects of its overexpression. 2 treatment groups are presented. In the first, TDP-43 knockout (G2) is compared to control (A1) and rescue (G2; TDP-43-GAL4>UAS-TDP-43). In the second comparison, the motor neuron driver D42-GAL4 is used to overexpress TDP-43 in motor neurons, using LacZ as a control for overexpression of a foreign protein.

2012-06-30 | E-GEOD-31194 | biostudies-arrayexpress

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