Studies

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22 Studies visible to you, out of a total of 24

The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry that is suitable to specifically map the content of autophagosomes and potentially other transient vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched ...

Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFβ-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFβ. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. ...

Hexanucleotide repeat expansions within C9orf72 are a frequent cause of amyotrophic lateral sclerosis and frontotemporal dementia. Haploinsufficiency leading to reduced C9orf72 protein contributes to disease pathogenesis. C9orf72 binds SMCR8 to form a robust complex that regulates small GTPases, lysosomal integrity, and autophagy. In contrast to this functional understanding, we know far less about the assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ...

Autophagy is responsible for degradation of an extensive portfolio of cytosolic cargoes that are engulfed in autophagosomes to facilitate their transport to lysosomes. Besides basal autophagy, which constantly degrades cellular material, the pathway is dynamically altered by different conditions, resulting in enhanced autophagosome formation and cargo turnover. The extensive profile of autophagosome content as well as the phospholipid composition of human autophagosome membranes remains elusive. ...

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, ...

The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6–59 amino-acids-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear. Here, we delineated the route of TAPL to lysosomes and investigated the determinants ...

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 ...

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