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

ABSTRACT: 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.

INSTRUMENT(S): autoflex

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Osteosarcoma

SUBMITTER: Yoshinori Takahashi

LAB HEAD: Yoshinori Takahashi

PROVIDER: PXD057301 | Pride | 2024-12-03

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	AP1.mzML	Mzml
	AP1_Slot1-37_1_2332.d.zip	Other
	AP1_Slot1-37_1_2332.mzid.gz	Mzid
	AP1_Slot1-37_1_2332.mzid_AP1_Slot1-37_1_2332.MGF	Mzid
	AP2.mzML	Mzml

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1 - 5 of 24

Publications

Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain.

Hamamoto Kouta K Liang Xinwen X Ito Ayako A Lanza Matthew M Bui Van V Zhang Jiawen J Opozda David M DM Hattori Tatsuya T Chen Longgui L Haddock David D Imamura Fumiaki F Wang Hong-Gang HG Takahashi Yoshinori Y

Cell reports 20241127 12

Macroautophagy (autophagy) involves the formation of phagophores that mature into autophagosomes. The impact of inhibiting autophagosome closure remains unclear. Here, we report the generation and analysis of mice with impaired autophagosome closure by targeting the ubiquitin E2 variant-like (UEVL) β strands of the endosomal sorting complex required for transport (ESCRT) I subunit VPS37A. The VPS37A UEVL mutation (Δ43-139) impairs bulk autophagic flux without disrupting ESCRT-I complex assembly ...[more]

PMID: 39607828

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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

Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002

Project description:Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the structure-function relationships between the intracellular structures of cyanobacteria and their roles in cells. While these studies have made great progress in understanding cyanobacterial physiology, the previous fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We have performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including PSII assembly in cyanobacteria with enhanced spatiotemporal resolution.

2021-09-09 | PXD021787 | Pride

RBM6 proximity interactions (RBM6-APEX)

Project description:We sought to map RBM6 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. Toward this end, we used CRISPR-cas9 methodology to establish MCF10A cell line expressing APEX2 fused to the N-terminal of RBM6, hereafter called MCF10AAPEX2-RBM6. The peroxidase activity of APEX2-RBM6 fusion was confirmed. Next, RBM6 proximal proteins that were biotinylated by APEX2 activation using hydrogen peroxidase (H2O2) in the presence of biotin-phenol were isolated and subjected to mass spectrometry. We identified 173 (P-value<0.05) RBM6 proximity-interaction partners.

2021-12-29 | PXD024295 | 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

SILAC APEX2-GAL9 upon LLOMe or GPN treatment

Project description:Proximity proteomics of GAL9 upon lysosomal damage by LLOMe or GPN. Proximity labeling was performed in SILAC labelled HEK293T cells stably expressing APEX2-GAL9.

2020-01-30 | PXD015779 | Pride

Proximity labeling by a recombinant APEX2-FGF1 fusion protein reveals interaction of FGF1 with the proteoglycans CD44 and CSPG4

Project description:Fibroblast growth factor 1 (FGF1) binds to specific FGF receptors (FGFRs) at the surface of target cells to initiate intracellular signaling. In addition, FGF1 also binds to heparan sulfate proteoglycans (HSPG), which act as important co-receptors. Even if some interactions with HSPGs have been characterized, it is not entirely clear if FGF1 could interact with additional proteoglycans at the cell surface. We have devised and tested a method to identify novel binding sites for FGF1 at the cell surface, which may also be applicable for other protein ligands. First, we constructed an APEX2-FGF1 fusion protein to perform proximal biotin labeling of proteins after binding of the fusion protein to cells. After functional validation of the construct, we used this method to identify binding sites for FGF1 on living cells. We confirmed the feasibility of our approach by easy detection of FGFR4, a well-known and specific receptor for FGF1. We then performed a screen in RPE1 cells and among the top hits were the proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44 through its heparin-binding moiety. Moreover, we found that FGF1 co-localizes with both CSPG4 and CD44 at the cell surface suggesting that these receptors act as storage molecules creating a reservoir of FGF1. Importantly, our data demonstrate that recombinant ligand-APEX2 fusion proteins can be used to identify novel receptor interactions at the cell-surface.

2018-06-06 | PXD009302 | Pride

Autophagy content profiling in human cells

Project description:Identification of autophagy substrates by directing APEX2 to autophagosomes and immune isolation of lysosomes.

2021-01-27 | PXD020758 | Pride

Charecterization of RBM42 interacting proteins in HCT116 cells with and without DNA damage

Project description:RNA binding motif protein 42 (RBM42) is an understudied gene mapped to 19q13.12 region and codes for a protein that consists of 480 amino acids, containing one RNA recognition motif (RRM) at its C-terminal region. We mapped the RBM42 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. We utilized CRISPR-Cas9 methodology for biallelic knock-in APEX2 at the C-terminus of the endogenous RBM42 coding sequence (CDS) and established an HCT116 cell line expressing RBM42-APEX2 fusion. RBM42 interactome mapping was performed by using Control and and HCT116 expressing RBM42-APEX2 cells that were treated with DMSO or Etoposide (VP-16) that induce DNA repair and incubated in media containing biotin phenol, followed by H2O2 treatment, to activate APEX2 peroxidase activity. Pathway enrichment analysis revealed that the majority of proteins that appeared in proximity to RBM42 are implicated in mRNA splicing and processing. Furthermore, we observed a significant enrichment of proteins involved in cell cycle checkpoints and regulation, further highlighting the role of RBM42 in DNA damage response. Together, RBM42 interactome analysis substantiates its function as a splicing regulator and implicates it in additional cellular pathways related to mRNA metabolism and DNA damage response.

2023-11-22 | PXD034854 | Pride

A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for DNAJC13 in opioid receptor downregulation

Project description:G protein-coupled receptor (GPCR) activity is critically regulated by trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). Using our GPCR-APEX2/AUR pipeline, we demonstrate that a gene identified in our screen, DNAJC13, plays a conserved role in the trafficking of activated DOR to the lysosome. To determine how depletion of DNAJC13 changes the local proteome of DOR-positive endosomes in cultured cells, we made cell lines stably expressing DOR-APEX2 or GFP-APEX2 (cytoplasmic). We then used siRNAs to knockdown DNAJC13 from DOR-APEX2 cells and compared these results to a siRNA control. Three independent biological replicates of the proximity labeling reaction were performed follow by purification of the biotinylated proteins, trypsinization, purification of peptides, TMT labeling, and detection and analysis by MS. In addition, this TMT-dataset (18-plex) contains 6 additional samples of from an independent experiment analyzing the proximity labeling of a 2xFYVE-GFP-APEX2 construct, but these samples were not included in further data analysis. We found that the DOR proximal proteome was highly enriched in endosomal proteins compared to the cytoplasmic control, and found 13 proteins significantly changed (FDR<0.1) with DNAJC13 knockdown.

2024-09-03 | PXD044921 | 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).

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