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Proteolysis of zyxin by HtrA1 induces nuclear translocation of the resulting C-terminal fragment of zyxin

ABSTRACT: We identified two neo-N termini derived from proteolytic cleavage of the mechanotransducer zyxin at Asp149 and Val276. We correlated these proteolytic events with the activity of caspase-1 and serine protease HtrA1, respectively, and observed nuclear translocation of the fragment zyxin277-572. To evaluate the biological role of nuclear zyxin we used Tandem Mass Tags (TMT)-based quantitative proteomics to compare protein abundances in HeLa cell ectopically expressing zyxin277-572, full length zyxin and control cells.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Fabio Sabino

LAB HEAD: Ulrich auf dem Keller

PROVIDER: PXD017432 | Pride | 2020-09-07

REPOSITORIES: Pride

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			Action	DRS
	20181026_FJ_FS_HeLa_zyxin_10PLEX_28CE.msfView	Msf
	20181026_FJ_FS_HeLa_zyxin_10PLEX_28CE.pdResult	Other
	20181026_FJ_FS_HeLa_zyxin_10PLEX_28CE.pep.pep.xml	Pepxml
	20181026_FJ_FS_HeLa_zyxin_10PLEX_28CE.raw	Raw

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Publications

Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival.

Sabino Fabio F Madzharova Elizabeta E Auf dem Keller Ulrich U

Cell death & disease 20200821 8

Proteases modulate critical processes in cutaneous tissue repair to orchestrate inflammation, cell proliferation and tissue remodeling. However, the functional consequences and implications in healing impairments of most cleavage events are not understood. Using iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) we had characterized proteolytic signatures in a porcine wound healing model and identified two neo-N termini derived from proteolytic cleavage of the focal adhesion prot ...[more]

PMID: 32826880

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HtrA1-dependent proteolysis of TGF-β controls both neuronal maturation and developmental survival

Project description:Transforming growth factor-β (TGF-β) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-β accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice over-expressing a TGF-β responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-β signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-β1, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-β signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-β could be one of the critical events controlling both neuronal maturation and developmental survival. Keywords: HtrA1 / neuronal survival / PAI-1 / TGF-β signalling / tPA

2008-04-16 | GSE11162 | GEO

Comparative degradomics of porcine and human wound exudates unravels biomarker candidates for assessment of wound healing progression in trauma patients

Project description:Impaired cutaneous wound healing is a major complication in elderly people and patients suffering from diabetes with raising rates in industrialized countries. Heterogeneity of clinical manifestations hampers effective molecular diagnostics and decisions for appropriate therapeutic regimens. Using a customized positional quantitative proteomics workflow, we have established a time-resolved proteome and N-terminome resource from wound exudates in a clinical pig wound model that we exploited as robust template to interpret a heterogeneous dataset from patients undergoing the same wound treatment. With Zyxin, IQGAP1 and HtrA1, this analysis and validation by targeted proteomics identified differential abundances and proteolytic processing of proteins of epidermal and dermal origin as prospective biomarker candidates for assessment of critical turning points in wound progression. Thus, we demonstrate the power of a fine-tuned animal wound model to bridge the translational gap as prerequisite for future extended clinical studies with large cohorts of individuals affected by healing impairments.

2017-09-14 | PXD006674 | Pride

HtrA1-dependent proteolysis of TGF-M-NM-2 controls both neuronal maturation and developmental survival

Project description:Transforming growth factor-M-NM-2 (TGF-M-NM-2) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-M-NM-2 accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice over-expressing a TGF-M-NM-2 responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-M-NM-2 signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-M-NM-21, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-M-NM-2 signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-M-NM-2 could be one of the critical events controlling both neuronal maturation and developmental survival. Keywords: HtrA1 / neuronal survival / PAI-1 / TGF-M-NM-2 signalling / tPA Total RNA were extracted from 3 cultures of 2 DIV Human neurons. For each stage, equal amounts of each RNA were pooled and 5M-BM-5g were reverse-transcribed, labelled and hybridized on pangenomic microarrays. Each pool was hybridized in duplicate dye-swap independent experiments.

2008-04-15 | E-GEOD-11162 | biostudies-arrayexpress

Comparative transcriptome data of ARPE-19 cells with and without HTRA1 overexpression

Project description:To explore the key signaling pathways that might be affected by up-regulated HTRA1, we performed RNA-seq analysis on ARPE-19 cells infected with HTRA1 adenovirus and negative control adenovirus for 24 h. Compare with control groups, there were 2810 differentially expressed genes (log2 |fold change|> 1, log10 adjusted p-values < 0.05) were identified to be associated with distinct biological processes. 1053 genes were significantly upregulated and 1757 genes were significantly downregulated in ARPE-19 cells overexpressing HTRA1. The KEGG pathways analysis of up-regulated and down-regulated genes and ranking the enrichment pathways according to the corrected p-value. In the KEGG pathways analysis of up-regulated genes, the metabolic pathways ranked first with 84 genes involved. And in the KEGG pathways analysis of down-regulated genes, the metabolic pathways ranked third with 104 genes involved. These results revealed that up-regulated HTRA1 prominently disturbed the metabolic pathways in ARPE-19 cells.

2023-06-22 | GSE186816 | GEO

HTRA1 dependent cell cycle proteomics

Project description:The HTRA1 gene encoding an evolutionary conserved protein quality control factor can be epigenetically silenced or inactivated by mutation under pathologic conditions such as cancer. Recent evidence suggests that loss of HTRA1 function causes multiple phenotypes including acceleration of cell growth, delayed onset of senescence, centrosome amplification and polyploidy suggesting an implication in the regulation of the cell cycle. To address this model, we performed a large-scale proteomics study to correlate the abundance of proteins and HTRA1 levels in various cell cycle phases using label-free quantification mass spectrometry. These data indicate that the levels of 4723 proteins fluctuated in a cell cycle-dependent, 2872 in a HTRA1-dependent and 1530 in a cell cycle- and HTRA1-dependent manner. The large number of proteins affected by the modulation of HTRA1 levels support its general role in protein homeostasis. Moreover, the detected changes in protein abundance in combination with pull down data implicate HTRA1 is in numerous cell cycle events such as DNA replication, chromosome segregation and cell cycle dependent apoptosis. These results highlight the wide implications of HTRA1 in cellular physiology

2019-02-15 | PXD007079 | Pride

Recombinant TSP1 cleaved by HTRA1 (LCMS)

Project description:HTRA1 proteolysis of TSP1 inhibits immune suppression in age-related macular degeneration. A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age related macular degeneration (AMD), a major cause of blindness affecting millions of individuals worldwide. Here we show that monocytes (Mo) from homozygous carriers of the 10q26 AMD-risk haplotype overexpress the High-Temperature Requirement A Serine Peptidase 1 (HTRA1), which locates to mononuclear phagocytes (MP) in eyes of patients with AMD. Elevated HTRA1 led to significant subretinal Mo persistence and promoted pathogenic MP accumulation due to the hydrolysis of Thrombospondin 1 (TSP1). Mechanistically, we demonstrate that HTRA1 separates TSP1’s two CD47- binding Valine-Valine-Methionine-sites that are necessary for efficient CD47 activation and repression of osteopontin (OPN) secretion, a mediator of inflammation and wound healing. Accordingly, OPN was overexpressed in early Mo-derived macrophages in 10q26 risk carriers and OPN deletion or inhibition fully reversed HTRA1- induced pathogenic MP persistence. Our observations provide new insights into the molecular mechanisms of MP accumulation in inflammation and show that HTRA1 resistant CD47 agonists and OPN inhibitors can provide a powerful tool to reverse HTRA1’s pro-inflammatory effect and restore retinal immune suppression in AMD, critical for retinal homeostasis.

2020-07-25 | PXD020546 | Pride

Rational correction of pathogenic conformational defects in HTRA1

Project description:Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we show that disease-causing mutations targeting the protomer-protomer interface impair trimerization. Focusing on a prototypical interface mutation (R274Q), we designed an HTRA1 variant that complemented pathogenic HTRA1 and reconstituted its multimeric assembly and enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrated that expression of this protein-based corrector in trans was sufficient to stabilize HTRA1-R274Q and restore the cerebrovascular proteome. As an alternative approach to achieve repair of pathogenic interface mutants, we generated supramolecular chemical ligands that shifted the monomer-trimer equilibrium by stabilizing proteolytically active trimers. Moreover, we identified a peptidic ligand that activated HTRA1 monomers. Collectively, our findings open novel perspectives for tailored protein repair strategies.

2023-11-24 | PXD024683 | Pride

Proteome analysis of brain microvessels from htra1 KO mice

Project description:CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathyis) a small-vessel disease caused by loss of function mutaions of htrA1, which cleaves several extracellular matrix proteins. Here, we isolated microvessels from htra1 KO and wild type control mice to study the effect of htra1 loss of function on microvessels.

2018-08-03 | PXD009411 | Pride

Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1

Project description:Proteolytic destruction of articular cartilage is a major pathogenic mechanism in osteoarthritis (OA), but was not previously investigated on a proteome-wide scale. We sought to define the human knee OA cartilage degradome and the contribution therein of the serine protease HtrA1, using an N-terminomics strategy, terminal amine isotopic labeling of substrates (TAILS).

2023-11-03 | PXD028320 | Pride

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy

Project description:Genetic polymorphisms in the region of the trimeric serine hydrolase HTRA1 are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remains undefined. In this study, we have developed an HtrA1 blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in pre-clinical models to elucidate the in vivo repertoire of HtrA1 specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD that were treated with the HtrA1 blocking Fab fragment. To our knowledge, this is the first pharmacodynamic biomarker measuring ocular protease activity in AMD.

2020-01-22 | MSV000084831 | MassIVE

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