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65 Publications visible to you, out of a total of 65
Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling.

Abstract (Expand)

Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways … exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis-challenging conditions in human cell lines. We identified various autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow dissection of the proteostasis contribution of autophagy to numerous individual proteins.

Authors: Susanne Zellner, Martina Schifferer, Christian Behrends

Date Published: 18th Mar 2021

Publication Type: Journal

CRISPR-Mediated Induction of Neuron-Enriched Mitochondrial Proteins Boosts Direct Glia-to-Neuron Conversion.

Abstract (Expand)

Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is … unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion.

Authors: Gianluca L Russo, Giovanna Sonsalla, Poornemaa Natarajan, Christopher T Breunig, Giorgia Bulli, Juliane Merl-Pham, Sabine Schmitt, Jessica Giehrl-Schwab, Florian Giesert, Martin Jastroch, Hans Zischka, Wolfgang Wurst, Stefan H Stricker, Stefanie M Hauck, Giacomo Masserdotti, Magdalena Götz

Date Published: 4th Mar 2021

Publication Type: Journal

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.

Abstract (Expand)

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent … pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1(-/-) microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.

Authors: A. Colombo, L. Dinkel, S. A. Muller, L. Sebastian Monasor, M. Schifferer, L. Cantuti-Castelvetri, J. Konig, L. Vidatic, T. Bremova-Ertl, A. P. Lieberman, S. Hecimovic, M. Simons, S. F. Lichtenthaler, M. Strupp, S. A. Schneider, S. Tahirovic

Date Published: 24th Feb 2021

Publication Type: Journal

An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome.

Abstract (Expand)

To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we … miniaturized secretome analysis by developing the "high-performance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.

Authors: J. Tushaus, S. A. Muller, E. S. Kataka, J. Zaucha, L. Sebastian Monasor, M. Su, G. Guner, G. Jocher, S. Tahirovic, D. Frishman, M. Simons, S. F. Lichtenthaler

Date Published: 15th Oct 2020

Publication Type: Journal

Neuronal Differentiation of LUHMES Cells Induces Substantial Changes of the Proteome.

Abstract (Expand)

Neuronal cell lines are important model systems to study mechanisms of neurodegenerative diseases. One example is the Lund Human Mesencephalic (LUHMES) cell line, which can differentiate into … dopaminergic-like neurons and is frequently used to study mechanisms of Parkinson's disease and neurotoxicity. Neuronal differentiation of LUHMES cells is commonly verified with selected neuronal markers, but little is known about the proteome-wide protein abundance changes during differentiation. Using mass spectrometry and label-free quantification (LFQ), the proteome of differentiated and undifferentiated LUHMES cells and of primary murine midbrain neurons are compared. Neuronal differentiation induced substantial changes of the LUHMES cell proteome, with proliferation-related proteins being strongly down-regulated and neuronal and dopaminergic proteins, such as L1CAM and alpha-synuclein (SNCA) being up to 1,000-fold up-regulated. Several of these proteins, including MAPT and SYN1, may be useful as new markers for experimentally validating neuronal differentiation of LUHMES cells. Primary midbrain neurons are slightly more closely related to differentiated than to undifferentiated LUHMES cells, in particular with respect to the abundance of proteins related to neurodegeneration. In summary, the analysis demonstrates that differentiated LUHMES cells are a suitable model for studies on neurodegeneration and provides a resource of the proteome-wide changes during neuronal differentiation. (ProteomeXchange identifier PXD020044).

Authors: J. Tushaus, E. S. Kataka, J. Zaucha, D. Frishman, S. A. Muller, S. F. Lichtenthaler

Date Published: 21st Sep 2020

Publication Type: Journal

ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis.

Abstract (Expand)

While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins … (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

Authors: F. Eck, S. Phuyal, M. D. Smith, M. Kaulich, S. Wilkinson, H. Farhan, C. Behrends

Date Published: 16th Sep 2020

Publication Type: Journal

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex.

Abstract (Expand)

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and … inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Authors: C. Z. Koo, N. Harrison, P. J. Noy, J. Szyroka, A. L. Matthews, H. E. Hsia, S. A. Muller, J. Tushaus, J. Goulding, K. Willis, C. Apicella, B. Cragoe, E. Davis, M. Keles, A. Malinova, T. A. McFarlane, P. R. Morrison, H. T. H. Nguyen, M. C. Sykes, H. Ahmed, A. Di Maio, L. Seipold, P. Saftig, E. Cull, C. Pliotas, E. Rubinstein, N. S. Poulter, S. J. Briddon, N. D. Holliday, S. F. Lichtenthaler, M. G. Tomlinson

Date Published: 4th Sep 2020

Publication Type: Journal

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