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22 Publications visible to you, out of a total of 22
Lipid and protein content profiling of isolated native autophagic vesicles.

Abstract (Expand)

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is … highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.

Authors: D. Schmitt, S. Bozkurt, P. Henning-Domres, H. Huesmann, S. Eimer, L. Bindila, C. Behrends, E. Boyle, F. Wilfling, G. Tascher, C. Munch, C. Behl, A. Kern

Date Published: 6th Dec 2022

Publication Type: Journal

ALS-linked loss of Cyclin-F function affects HSP90.

Abstract (Expand)

The founding member of the F-box protein family, Cyclin-F, serves as a substrate adaptor for the E3 ligase Skp1-Cul1-F-box (SCF)<sup>Cyclin-F</sup> which is responsible for … ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in <i>CCNF</i> encoding for Cyclin-F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether <i>CCNF</i> mutations affect the substrate adaptor function of Cyclin-F and whether altered SCF<sup>Cyclin-F</sup>-mediated ubiquitination contributes to pathogenesis in <i>CCNF</i> mutation carriers. To address these questions, we set out to identify new SCF<sup>Cyclin-F</sup> targets in neuronal and ALS patient-derived cells. Mass spectrometry-based ubiquitinome profiling of <i>CCNF</i> knockout and mutant cell lines as well as Cyclin-F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses provided evidence for a Cyclin-F-dependent association and ubiquitination of HSP90AB1 and implied a regulatory role that could affect the binding of a number of HSP90 clients and co-factors. Together, our results point to a possible Cyclin-F loss-of-function-mediated chaperone dysregulation that might be relevant for ALS.

Authors: A. Siebert, V. Gattringer, J. H. Weishaupt, C. Behrends

Date Published: 16th Sep 2022

Publication Type: Journal

Mapping autophagosome contents identifies interleukin-7 receptor-alpha as a key cargo modulating CD4+ T cell proliferation.

Abstract (Expand)

CD4+ T cells are pivotal cells playing roles in the orchestration of humoral and cytotoxic immune responses. It is known that CD4+ T cell proliferation relies on autophagy, but identification of the … autophagosomal cargo involved is missing. Here we create a transgenic mouse model, to enable direct mapping of the proteinaceous content of autophagosomes in primary cells by LC3 proximity labelling. Interleukin-7 receptor-alpha, a cytokine receptor mostly found in naive and memory T cells, is reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy show increased interleukin-7 receptor-alpha surface expression, while no defect in internalisation is observed. Mechanistically, excessive surface interleukin-7 receptor-alpha sequestrates the common gamma chain, impairing the interleukin-2 receptor assembly and downstream signalling crucial for T cell proliferation. This study shows that key autophagy substrates can be reliably identified in this mouse model and help mechanistically unravel autophagy's contribution to healthy physiology and disease.

Authors: D. Zhou, M. Borsa, D. J. Puleston, S. Zellner, J. Capera, S. Sanderson, M. Schifferer, S. S. Hester, X. Ge, R. Fischer, L. Jostins, C. Behrends, G. Alsaleh, A. K. Simon

Date Published: 2nd Sep 2022

Publication Type: Journal

Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

Abstract (Expand)

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 removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the 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.

Authors: B. Kravic, T. Bionda, A. Siebert, P. Gahlot, S. Levantovsky, C. Behrends, H. Meyer

Date Published: 21st Jul 2022

Publication Type: Journal

Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.

Abstract (Expand)

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 UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.

Authors: L. Strohm, Z. Hu, Y. Suk, A. Ruhmkorf, E. Sternburg, V. Gattringer, H. Riemenschneider, R. Berutti, E. Graf, J. H. Weishaupt, M. S. Brill, A. B. Harbauer, D. Dormann, J. Dengjel, D. Edbauer, C. Behrends

Date Published: 1st Jul 2022

Publication Type: Journal

Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells.

Abstract (Expand)

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, suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified. For complete details on the use and execution of this protocol, please refer to Zellner et al. (2021).

Authors: S. Zellner, K. Nalbach, C. Behrends

Date Published: 18th Jun 2021

Publication Type: Journal

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

Abstract (Expand)

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, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Authors: T. N. Nguyen, B. S. Padman, S. Zellner, G. Khuu, L. Uoselis, W. K. Lam, M. Skulsuppaisarn, R. S. J. Lindblom, E. M. Watts, C. Behrends, M. Lazarou

Date Published: 6th May 2021

Publication Type: Journal

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