Autophagosome content profiling in TBK1-E696K knockin MEFs
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Embryo, Fibroblast
DISEASE(S): Huntington Disease
SUBMITTER: 
Christian Behrends
LAB HEAD: Christian Behrends
PROVIDER: PXD048795 | Pride | 2024-03-16
REPOSITORIES: Pride
Similar Datasets
Project description:Identification of autophagy substrates by directing APEX2 to autophagosomes and immune isolation of lysosomes.
2021-01-27 | PXD020758 | 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: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
Project description:SILAC-based proximity proteomics of UAPEX2-tagged UBE2QL1 in differential LLOMe-treated HeLa cells
2019-08-22 | PXD014521 | Pride
Project description:Autophagy has been implicated as a host defense mechanism against intracellular pathogens. However, certain intracellular pathogens such as Leishmania can manipulate the host’s autophagy to promote their survival. Recently, our findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of mammalian target of rapamycin complex 1 regulation. This occurs in the background of enhanced mTOR activity, which suggests the fine-tuning of autophagy to optimally promote parasite survival and may involve the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Select proteomics results were validated by immunoblotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level.
2023-05-10 | PXD037057 | Pride
Project description:Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been studied extensively, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. To start filling these gaps, we devised a differential centrifugation set up, where we enriched intact autophagosomes and performed affinity purification-mass spectrometry (AP-MS), using ATG8 as a bait. This method enabled the identification of an autophagy adaptor protein.
2022-10-18 | PXD031787 | Pride
Project description:LC-MS/MS analysis for protein quantification of mouse brain and liver tissues
2021-05-18 | PXD025201 | Pride
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:The Tau (MAPT) protein drives neuronal dysfunction and toxicity in the brain in Alzheimer’s disease (AD) and other Tauopathies. To dissect the complexity of the Tau interactome that underlies this process we used two proteomic approaches to characterize the dynamic and multifaceted nature of Tau protein-protein interactions in human induced pluripotent stem cell (iPSC)-derived neurons. We used engineered ascorbic acid peroxidase (APEX) for spatiotemporally restricted mapping of Tau interaction proteins in combination with quantitative affinity purification mass spectrometry (AP-MS) to interrogate disease-related changes in the Tau interactome. The APEX method resolved subcellular interactions of wild-type Tau at amino acid level resolution in living neurons as well as novel activity-dependent interactions of Tau with presynaptic vesicle proteins that occurred during Tau secretion from neurons. Among the many Tau interacting proteins revealed by AP-MS, the interaction of mitochondrial proteins with wild-type Tau (TauWT) was more robust than with TauV337M or TauP301L. The mitochondrial proteins that preferentially interacted with TauWT comprised a protein module that is downregulated in multi-omics analyses of human AD brains. Mitochondrial bioenergetics were altered in TauV337M compared to TauWT human iPSC-derived neurons confirming the impact of Tau on mitochondria. These Tau interactome analyses open up novel disease-related processes as potential therapeutic targets to block Tau-mediated pathogenesis.
2022-01-11 | PXD026306 | Pride
Project description:Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosomal proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both Hela cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.
2021-09-23 | PXD027476 | Pride