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AEP targets HEK293 cells using specific inhibitor and TurboID approaches

ABSTRACT: Characterisation of AEP physiological targets using specific inhibitors and TurboID approaches

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Jonathan Martinez Fabregas

LAB HEAD: Jonathan Martinez Fabregas

PROVIDER: PXD053016 | Pride | 2025-05-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	AEP_TiD_1.raw	Raw
	AEP_TiD_2.raw	Raw
	AEP_TiD_3.raw	Raw
	AEP_TiD_proteinGroups.txt	Txt
	HEK293_Control_vs_MVO_peptides.txt	Txt

Items per page:

1 - 5 of 18

Publications

Radiotherapy resistance driven by Asparagine endopeptidase through ATR pathway modulation in breast cancer.

Morillo-Huesca Macarena M G López-Cepero Ignacio I Conesa-Bakkali Ryan R Tomé Mercedes M Watts Colin C Huertas Pablo P Moreno-Bueno Gema G Durán Raúl V RV Martínez-Fábregas Jonathan J

Journal of experimental & clinical cancer research : CR 20250227 1

<h4>Background</h4>Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear.<h4>Methods</h4>High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients ...[more]

PMID: 40012043

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Proteomics- and BRET-screens identify SPRY2 as interactor of active Ras the impacts on its membrane organisation

Project description:K-Ras is regulated and active in nanoscale proteo-lipid domains of the plasma membrane. Few regulators of K-Ras membrane organisation have been identified so far. We here combined a primary TurboID-based proximal proteome screen with a secondary BRET-screen to identify nine novel bona fide interactors of the K-Ras G-domain that would modulate its membrane organisation. We focused our hit characterisation on one novel candidate, APLP2, and SPRY2, which was previously implicated as negative regulator of MAPK-signalling. While APLP2 appeared to indirectly bind to K-Ras via C-Raf, SPRY2 showed characteristics of a new effector protein. We show by co-immunoprecipitation and BRET-experiments that the C-terminal fragment of SPRY2 comprising residues 161-315 interacts more with K-RasG12V than the full-length protein. Both full length and C-terminal fragment localized to the membrane. SPRY2 plasma membrane localization was blocked if K-Ras membrane anchorage was inhibited. Likewise, binding to oncogenic K-Ras was disrupted by K-Ras-inhibitors. Mutations at the predicted interface of the K-Ras effector binding region and the C-terminal fragment of SPRY2 modulated the interaction. Our data further suggest that SPRY2 operates as homo- or hetero-di/oligomer with SPRY4. Both full length SPRY2 and its C-terminal fragment promote differentiation of muscle C2C12 cells, which requires inhibition of MAPK-signalling. We propose a model, wherein active K-Ras recruits SPRY-dimers via the C-terminus of SPRY2 to the plasma membrane where they bind directly to Ras and block access of effectors.

2025-10-02 | PXD067415 | Pride

Understanding the physiological role of asparaginyl endopeptidase (AEP)/Legumain

Project description:Understanding the regulation of lysosomal proteolytic/hidrolytic capacity has been recently advanced in a huge manner by the discovery of the lysosome-mTOR-TFEB pathway connecting nutrient sensing, autophagy and de novo lysosome generation. But, how lysosomes can adjust their proteolytic/hidrolytic capacity to meet varying conditions (more or less substrate) under normal fed conditions is not known. We have used AEP as a proxy to understand how lysosomes can compensate the lack of a key lysosomal protease to meet the requirements under physiological, fed conditions. To do this, we did compare cytoplasmic, membrane and nuclear fractions of WT and AEP MEFs that were expanded in SILAC media. AEP knock-out mice show pale kidney, with abnormal proliferation of the epithelial proximal tubular cells. In an attempt to understand the role of AEP in the kidney, we did compare kidney obtained from WT and AEP mice using kidneys from WT mice that were fed with SILAC food. Also, in an attempt to reproduce the changes observed in the absence of AEP in kidney and MEFs, we treated WT MEFs grown in SILAC media with MVO26630, specific inhibitor of AEP, for 12 and 48 hours.

2019-01-09 | PXD011473 | Pride

Endothelial C/EBPβ/AEP Pathway Drives Vascular Deterioration and Systemic Aging

Project description:Age-related microvascular dysfunction disrupts nutrient homeostasis and waste clearance, leading to organ failure. However, a critical gap remains in our understanding of the specific molecular drivers of vascular deterioration and how they orchestrate organism-wide aging. Here, we identify progressive activation of the CCAAT/enhancer-binding protein β (C/EBPβ)/asparagine endopeptidase (AEP) pathway in aging vascular endothelial cells contributes to vascular degeneration and lifespan reduction. Endothelial-specific C/EBPβ or AEP overexpression accelerated vascular aging and shortened lifespan in mice. Mechanistically, AEP mediates proteolytic cleavage of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (oxidised form, NAD+) biosynthesis, resulting in systemic NAD⁺ depletion and senescence that elicits both central and peripheral vascular dysfunction and ultimately systemic aging. Genetic ablation of AEP or expression of AEP-resistant NAMPT N136A mutant significantly ameliorated vascular aging and extended lifespan in endothelial-specific Tie 2-C/EBPβ transgenic mice. Pharmacologically, AEP inhibitor CP#11A or nicotinamide mononucleotide (NMN) supplementation alleviated age-related vascular decline, with CP#11A showing superior efficacy. These findings support a model in which endothelial senescence contributes to vascular dysfunction and systemic aging mediated by C/EBPβ/AEP signaling, and establish C/EBPβ/AEP as a therapeutic target to preserve vascular integrity and mitigate systemic frailty in aging populations.

2026-05-30 | E-MTAB-17006 | biostudies-arrayexpress

NPAS3-HEK293-standardcond-Illumina

Project description:NPAS3 or truncated NPAS3 overexpressed HEK293 cells or control HEK293 cells were collected at 24 hours after gene overexpression.Total RNA from alll samples was extracted.Biotinylated cRNA was prepared using the Illumina total Prep RNA application Kits.Hybridization, washing and scanning were performed according to the Illumina BeadStation 500* manual (revision C)

2011-10-03 | E-TABM-1078 | biostudies-arrayexpress

NPAS3-HEK293-circadian-Illumina

Project description:The fragments of FLNPAS3 and DeltaNPAS3 were transferred into a similar TET-inducible expression plasmid and transfected into HEK293 [T-REx-293] cells (Invitrogen). At the zero hour time-point, cells were shifted into a serum-rich medium plus tetracycline in order to induce circadian cycling and NPAS3 overexpression. Parental HEK293 [T-REx-293] cells after circadian rhythmcity induction for +12hr/+24hrs were used as negative controls because stably integrated FLNPAS3/DeltaNPAS3 cell lines showed a low level of leaky transcription in the absence of tetracycline. At +2 hours, cells were washed with DMEM and then incubated with the same plus tetracycline for the remaining period of the experiment. Cell samples were collected at +12hrs and +24hrs respectively. For all circadian cell culture experiments with FLNPAS3/DeltaNPAS3/parental negative control cell lines, duplicate biological samples were assessed. Microarray probes synthesised from RNA extraction products was quantified using an Agilent Bioanalyzer to ensure high quality probes of equal quantity between experiments. Sentrix® HumanRef-8 v2 chips were used to detect gene expression profiles.

2011-10-03 | E-TABM-1079 | biostudies-arrayexpress

Mapping the interactome of OSCC prognostic-associated proteins NDRG1 and PGK1 through proximity labeling using TurboID

Project description:Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer, accounting for more than 90% of all oral malignancies worldwide. The identification of diagnostic and prognostic markers for OSCC is crucial for improving patient outcomes, as early detection and treatment are critical for successful management of this disease. In this regard, our group recently demonstrated that N-myc downstream-regulated gene 1 (NDRG1) and phosphoglycerate kinase 1 (PGK1) are prognostic markers in OSCC, however, little is known about the role of these proteins in OSCC development. To better understand their signaling pathways, we used TurboID-based proximity labeling to identify the interactomes of NDRG1 and PGK1 in HEK293 cells. Here, protein abundance patterns associated with three distinct time-points were utilized for protein clustering, allowing the identification of protein clusters with a “fast” or “slow” response to biotin. Functional enrichment of GO terms revealed processes mostly associated with mRNA processing for both PGK1 and NDRG1 “fast” clusters, while GO processes related to protein localization and catalytic activity were identified in PGK1 and NDRG1 “slow” clusters, respectively. A total of 65 out of 361 proteins from different clusters were also identified in neoplastic islands of OSCC tissues from our previous study. Additionally, 28 of these proteins have their gene expression associated with prognostic features such as death, metastasis and/or relapse in OSCC patients. Ultimately, our data enabled the identification of 17 previously known physical interactions and functional associations among these proteins, as well as 30 new putative interactions, characterizing a prognostic-associated interactome composed of 28 proteins. This interactome enables the prioritization of candidates that can be further explored in OSCC progression.

2025-05-06 | PXD048046 | Pride

Transcriptome of HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

Project description:Comparison of gene expression profile of HEK293-CT cells and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1) We used micrarrays to identify the repertoire of genes differentially expressed upon overexpression of the chromatin repressor BAHD1 in HEK293 cells.

2015-12-11 | E-GEOD-51863 | biostudies-arrayexpress

Methylated DNA immunoprecipitation-sequencing of HEK293 cells (HEK293-CT) and HEK293 cells stably over-expressing the BAHD1 gene (HEK-BAHD1)

Project description:we report the partial methylome (CG-rich regions) of HEK293 cells and HEK293 cells over-expressing the BAHD1 gene (HEK-BAHD1) We used MEDIP-seq to identify genomic regions differentially methylated upon overexpression of the chromatin repressor BAHD1 in HEK293 cells.

2015-12-11 | E-GEOD-51864 | biostudies-arrayexpress

Chromatin immunoprecipitation-sequencing of HEK293 cells (HEK293-HPT-blue) and HEK293 cells stably over-expressing the BAHD1 gene (HEK293-HPT-BAHD1)

Project description:We report the genomic occupancy of the BAHD1 protein in HEK293 cells over-expressing the BAHD1 gene (HEK293-HPT-BAHD1) We used Native ChIP-seq to identify DNA regions bound to BAHD1 (native ChIP involves use of native chromatin before precipitation of immune complexes, without formaldehyde crosslinking of proteins with nucleic acids)

2015-12-11 | E-GEOD-53372 | biostudies-arrayexpress

HEK293 CD63 CRISPR cell-derived small EVs

Project description:The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In this study, we assemble a global scope of CD63-dependent protein trafficking into small secreted vesicles.

2018-10-24 | PXD006514 | Pride

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