Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

APEX2 of BAX, BAK and DRP1 in healthy and apoptotic cells

ABSTRACT: To identify components of mitochondrial apoptotic foci, Flag-APEX2-tagged constructs targeting Bax, Bak, and Drp1 were generated. These constructs are designed to localize to foci at the mitochondrial outer membrane (MOM) during apoptosis. Following biotinylation with BP and H2O2, quantitative mass spectrometry was used to identify neighbouring proteins. Notably, the mitochondrial outer membrane (MOM) contains porins that allow the free diffusion of molecules, potentially resulting in the biotinylation of intermembrane space (IMS) and mitochondrial inner membrane (MIM)-resident proteins. To comprehensively analyse the protein composition of apoptotic foci, three independent quantitative mass spectrometry experiments were conducted. Each experiment involved comparing untreated (control) cells with apoptotic cells induced by staurosporine treatment. By utilizing APEX2-tagged constructs in live cells, the subcellular localization of proteins under both healthy and apoptotic conditions was preserved. Moreover, consistent biotinylation patterns were observed across all three constructs, validating the reliability of the approach for studying dynamic protein complexes in apoptosis.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Mirita Franz-Wachtel

LAB HEAD: Ana J. Garcia-Saez

PROVIDER: PXD055201 | Pride | 2026-03-27

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20170301_CO_0673AnGS_R09R10R11.raw	Raw
	20170427_CO_0673AnGS_DM_Mix1_R12R13R14_2.raw	Raw
	20170427_CO_0673AnGS_DM_Mix2_R15R16R17_2.raw	Raw
	checksum.txt	Txt
	search_R09R10R11.zip	Other

Items per page:

1 - 5 of 6

Similar Datasets

Proximity proteomics of phagophore-associated proteins using APEX2-LC3B in VPS37A KO U-2 OS cells

Project description:We utilized an APEX2-mediated proximity biotinylation approach to label proteins on LC3-positive membranes that accumulate in VPS37A-knockout (KO) U-2 OS cells. AP, APEX2 control; WT, APEX2-LC3B.

2024-12-03 | PXD057301 | Pride

APEX proximity labeling of murine NDUFS4 interacting proteins in podocytes

Project description:In this project we used an unbiased approach to identify the proteins that interact with NDUFS4 in the mouse podocytes using the APEX2 proximity labeling system. An immortalized murine podocyte was permanently transduced with a doxycycline (DOX) inducible NDUFS4-APEX2 chimeric construct. The cells were treated with DOX for 72 h to induce NDUFS4-APEX2 expression, followed by H2O2 activation of APEX2 mediated biotinylation of neighboring proteins. Biotinylated proteins were pull down by streptavidin beads and analyzed by LC-MS/MS. Cells without DOX induction or with DOX but without H2O2 activation were used as controls

2024-02-08 | PXD041378 | Pride

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-dependent Mitophagy

Project description:Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on mitochondrial outer membrane (MOM). While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages of the process. Here we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome and phosphoproteome during 18 hours after induction of mitophagy by mitochondrial uncoupler CCCP. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise, “outside-in” directed degradation of mitochondrial subcompartments. While ubiquitylation of MOM proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix and cytosolic proteins were also found ubiquitinated at later stages of mitophagy. Phosphoproteome analysis revealed a possible cross-talk between phosphorylation and ubiquitylation during mitophagy on several key parkin targets, such as VDAC1/2.

2022-01-17 | PXD027586 | Pride

Proximity labeling reveals differential interaction partners of the human mitochondrial import receptor proteins TOMM20 and TOMM70

Project description:Import of most mitochondrial proteins requires that their precursor proteins are bound by the (peripheral) receptor proteins Tom20, Tom22, and Tom70. For budding yeast Tom20 and Tom70, there is evidence of specific yet overlapping substrate recognition, but no such data is available for metazoan cells. Using APEX2-based proximity labeling, we thus created association profiles for human TOMM20 and TOMM70 in HeLa cells. We particularly focused on their interaction with RNA-binding proteins (RBPs) since there is evidence for RNA association with the mitochondrial outer membrane (MOM) and local translation at the mitochondrial surface, but these processes are poorly understood. Our results show a preferred association of several RBPs and translation factors with TOMM20 over TOMM70. These include SYNJBP2, a previously identified membrane-bound RBP that binds and protects mRNAs encoding mitochondrial proteins, and PAIP1, a translational co-activator interacting with polyA-binding protein. Translational inhibition by puromycin resulted in an even increased association of these RBPs with TOMM20 compared to TOMM70, suggesting that TOMM20 but not TOMM70 might play a role in preserving cellular hemostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM.

2026-01-07 | PXD057097 | Pride

Lysosomal damage proteomics in HeLa cells

Project description:To identify ubiquitylation targets following lysosomal damage in HeLa cells treated with LLOMe we performed quantitative ubiquitin-remmnant (diGly) profilig coupled to mass spectrometry (MS). In addition, we performed APEX2-based proximity biotinylation followed by MS analysis to identify proximity partners of one of the ubiquitylation targets (CNN2).

2022-06-23 | PXD029926 | Pride

APEX2 mediated proximity labeling to investigate the proximal proteome of mitochondria tethered ULK1 in wild type and fip200 knock down cells

Project description:The object of this project is to investigate the proximal proteome of ULK1 during mitophagy and the effects of a FIP200 knock down using the APEX2-mediated proximity labeling system. APEX2-ULK1 or APEX2 was fused to 2xFKBP-myc and stably expressed in a cell line stably expressing a truncated FIS1-FRB fusion protein. Treatment with rapalog induces the stable interaction between FRB and FKBP, resulting in tethering of FKBPx2-myc-APEX2-ULK1 or FKBPx2-myc-APEX2 to the outer mitochondrial membrane.

2024-10-12 | PXD047277 | Pride

Whole genome pooled shRNA screen to characterize Raptinal-induced apoptosis in the MIA PaCa-2 cell line

Project description:The goal of the study was to characterize the mechanism by which Raptinal induces apoptosis using a genome-wide pooled shRNA screen. Following drug treatment, the abundance of the shRNA constructs were determined and compared between DMSO and Raptinal-treated samples. Constructs depleted in Raptinal samples versus DMSO control, are expected to potentiate Raptinal-induced apoptosis by down regulating anti-apoptotic proteins. In contrast, shRNA constructs enriched upon Raptinal treatment are expected to target pro-apoptotic proteins, whose down regulation confers protection from Raptinal-induced apoptosis. Follow up experiments were performed on enriched shRNAs in order to validate potentially novel pro-apoptotic proteins.

2018-02-27 | GSE66423 | GEO

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

Project description:Different brain cell types play distinct roles in brain development and disease via cellular mechanisms. Molecular characterization of these cellular mechanisms using cell type-specific approaches, particularly at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain pose several technical barriers. To overcome these, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. We generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under resting conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes, and the LPS effect was partly captured by analysis of the TurboID-NES-labeled proteome of BV2 cells.

2023-03-11 | PXD036744 | Pride

Autophagosome content profiling in TBK1-E696K knockin MEFs

Project description:To determine how the mutant TBK1-E696K protein impacts autophagosomes, we performed autophagosome content profiling using protease protection coupled APEX2 proximity proteomics of autophagosomes of homozygous TBK1-E696K knockin and wiltype mouse embryonic fibroblasts (MEFs) transfected with a APEX2-LC3 as previously described in Zellner et al. 2021 Molecular Cell.

2024-03-16 | PXD048795 | Pride

Total RNA Sequencing analysis of total RNA and RNA isolated from mitochondrial pellets in CSDE1-knockdown sensory neurons

Project description:CSDE1 is an RNA-binding protein that associates with a subset of mitochondria-related transcripts. We previously observed that RNAs encoding mitochondrial proteins localized to the mitochondrial inner membrane (MIM), intermembrane space (IMS), matrix, and outer membrane (MOM) are enriched within the CSDE1-bound RNA pool. To determine whether CSDE1 regulates the abundance and mitochondrial localization of mitochondria-associated RNAs in sensory neurons, we performed RNA sequencing of both total RNA and RNA extracted from isolated mitochondria isolated from CSDE1-knockdown sensory neurons. CSDE1 knockdown was achieved using lentiviral transduction, and sequencing results were compared with control neurons infected with empty vector (pLKO).

2026-03-12 | GSE324285 | GEO