SILAC APEX2-GAL9 upon LLOMe or GPN treatment
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Early Embryonic Cell, Kidney
SUBMITTER: 
Christian Behrends
LAB HEAD: Christian Behrends
PROVIDER: PXD015779 | Pride | 2020-01-30
REPOSITORIES: Pride
Similar Datasets
Project description:Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and eliminated by VCP/p97 and proteasome for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in extraction of CNN2, and show that other membrane regulators including SNAREs, PICALM, AGFG1 and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.
2022-06-23 | PXD032903 | Pride
Project description:SILAC-based proximity proteomics of UAPEX2-tagged UBE2QL1 in differential LLOMe-treated HeLa cells
2019-08-22 | PXD014521 | Pride
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
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
Project description:CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naïve and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy’s contribution to healthy physiology and disease.
2022-07-08 | PXD033733 | Pride
Project description:Identification of autophagy substrates by directing APEX2 to autophagosomes and immune isolation of lysosomes.
2021-01-27 | PXD020758 | Pride
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
Project description:Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into stress granules (SGs) reduces translation, but it remains unclear whether SGs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress-adaptation in cancer. Transcripts enriched in PSs, defined by polysome fractionation and RNAseq, were compared with mRNAs complexed with the SG-nucleator protein, G3BP1, defined by spatially-restricted enzymatic tagging. Short-term oxidative stress profoundly altered mRNA translation by promoting selective enrichment of transcripts within SGs or PSs. G3BP1 participates in this compartmentalisation by sequestering transcripts in SGs. Under stress, G3BP1-bound transcripts are PS-depleted and encode proteins involved in mRNA translation, pro-apoptosis, and mitochondrial function. In contrast, specific PS-enriched transcripts disassociate from G3BP1 under stress to encode proteins involved in diverse cellular cytoprotective pathways. Therefore, G3BP1 partitioning guides selective translation to support stress adaptation and cell survival.
2020-04-30 | PXD015491 | Pride
Project description:Potential interacting proteins of Isthmin-1 in E11.5 intact tissue during early kidney development.
2023-05-11 | PXD040642 | Pride
Project description:In cells, several cargoes are delivered to the cell surface or the extracellular space via unconventional secretion routes. GRASP55 is a Golgi protein that regulates unconventional secretion of distinct proteins and controls the assembly and membrane stacking of Golgi cisternae. Recent work suggested that the role of GRASP55 in unconventional secretion may involve its relocalization to other organelles. However, the stimuli that drive GRASP55-dependent unconventional secretion, the signaling events that regulate GRASP55 function, the subcellular locations where GRASP55 acts, and the cargoes that follow this route for secretion remain unclear. Here, we show that mTORC1 directly phosphorylates GRASP55 at multiple sites to maintain its Golgi localization. Cellular stresses or drugs that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to autophagosomal / MVB structures. Using secretome and surfactome analyses in GRASP55-null cells, we identify for the first time numerous -previously unknown- cargoes that rely on this unconventional secretory pathway.
2021-09-09 | PXD020331 | Pride