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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Created: 9th Jul 2024 at 13:23
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Projects: SyNergy - published datasets
Institutions: DZNE
Projects: SyNergy - published datasets
Institutions: Klinikum der Universität München
Public web page: Not specified
Organisms: Mus musculus, Rattus norvegicus, Homo sapiens, Macaca mulatta, Sus scrofa
Submitter: Rainer Malik
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Assays: Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Affinity purification coupled with mass spectrometry proteomics, Bottom-up proteomics, Gel-based experiment, Proteomics analysis of brain endothelial cells, Proteomics of Inflammasome, SWATH MS, Shotgun Proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics, Shotgun proteomics
Snapshots: Snapshot 1
HeLa cells stably expressing CNN2-APEX2 were grown at 37°C in DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate. Cells were differentially treated with 5 µM NMS-873 for 15 min followed by 1 h 1 mM LLOMe (Sigma) and 2 h washout wihout any drugs. Proximity labeling was performed essentially as described before (Korver et al., 2019). Briefly, cells were incubated with 500 µM Biotin-Phenol during the last 30 min and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop ...
Submitter: Rainer Malik
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Proteomics
Study: CNN2/Lysosome damage
Organisms: Homo sapiens
SOPs: No SOPs
Data files: VCP-dependent CNN2 proximity proteomics
Snapshots: No snapshots
For diGly proteomics: HeLa cells were cultured in lysine- and arginine-free DMEM supplemented with dialyzed FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin/streptomycin and light (K0) lysine (38 μg/mL) and arginine (66 μg/ml). Medium and heavy media were the same except the light lysine was replaced with K4 and K8-lysine, respectively. Medium and heavy labeled cells were treated for 1 h with 250 µM LLOMe while light labeled were treated for 1 h with vehicle alone (EtOH). Light and heavy ...
Submitter: Rainer Malik
Assay type: Experimental Assay Type
Technology type: Technology Type
Investigation: Proteomics
Study: CNN2/Lysosome damage
Organisms: Homo sapiens
SOPs: No SOPs
Data files: Lysosomal damage proteomics in HeLa cells
Snapshots: No snapshots
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).
Creators: Rainer Malik, Christian Behrends
Submitter: Rainer Malik
Investigations: Proteomics
Studies: CNN2/Lysosome damage
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, ...
Creators: Rainer Malik, Christian Behrends
Submitter: Rainer Malik