Studies

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, ...

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 ...

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. ...

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, ...

The founding member of the F-box protein family, Cyclin F, serves as substrate adaptor for the Ubiquitin E3 ligase Skp1-Cul1-F-box (SCF)Cyclin F which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in CCNF encoding for Cyclin F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether CCNF mutations affect the substrate adaptor function of Cyclin F and whether altered SCFCyclin ...

Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD ...

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 ...