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52 Publications visible to you, out of a total of 52
Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Abstract (Expand)

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase … ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Authors: Matteo Calligaris, Donatella Pia Spanò, Maria Chiara Puccio, Stephan A Müller, Simone Bonelli, Margot Lo Pinto, Giovanni Zito, Carl P Blobel, Stefan F Lichtenthaler, Linda Troeberg, Simone Dario Scilabra

Date Published: 24th Sep 2024

Publication Type: Journal

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Abstract (Expand)

Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based … labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.

Authors: Urbi Mukhopadhyay, Sophie Levantovsky, Teresa Maria Carusone, Sarah Gharbi, Frank Stein, Christian Behrends, Sagar Bhogaraju

Date Published: 9th Aug 2024

Publication Type: Journal

Innate immune memory after brain injury drives inflammatory cardiac dysfunction.

Abstract (Expand)

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a … common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1β was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1β or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1β-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

Authors: Alba Simats, Sijia Zhang, Denise Messerer, Faye Chong, Sude Beşkardeş, Aparna Sharma Chivukula, Jiayu Cao, Simon Besson-Girard, Felipe A Montellano, Caroline Morbach, Olga Carofiglio, Alessio Ricci, Stefan Roth, Gemma Llovera, Rashween Singh, Yiming Chen, Severin Filser, Nikolaus Plesnila, Christian Braun, Hannah Spitzer, Özgün Gökçe, Martin Dichgans, Peter U Heuschmann, Kinta Hatakeyama, Eduardo Beltrán, Sebastian Clauss, Boyan Bonev, Christian Schulz, Arthur Liesz

Date Published: 22nd Jul 2024

Publication Type: Journal

Rational correction of pathogenic conformational defects in HTRA1.

Abstract (Expand)

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly … defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1<sup>R274Q</sup> mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

Authors: Nathalie Beaufort, Linda Ingendahl, Melisa Merdanovic, Andree Schmidt, David Podlesainski, Tim Richter, Thorben Neumann, Michael Kuszner, Ingrid R Vetter, Patricia Stege, Steven G Burston, Anto Filipovic, Yasser B Ruiz-Blanco, Kenny Bravo-Rodriguez, Joel Mieres-Perez, Christine Beuck, Stephan Uebel, Monika Zobawa, Jasmin Schillinger, Rainer Malik, Katalin Todorov-Völgyi, Juliana Rey, Annabell Roberti, Birte Hagemeier, Benedikt Wefers, Stephan A Müller, Wolfgang Wurst, Elsa Sanchez-Garcia, Alexander Zimmermann, Xiao-Yu Hu, Tim Clausen, Robert Huber, Stefan F Lichtenthaler, Carsten Schmuck, Michael Giese, Markus Kaiser, Michael Ehrmann, Martin Dichgans

Date Published: 16th Jul 2024

Publication Type: Journal

Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1.

Abstract (Expand)

Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In … this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.

Authors: Allwyn Pereira, Jeisimhan Diwakar, Giacomo Masserdotti, Sude Beşkardeş, Tatiana Simon, Younju So, Lucía Martín-Loarte, Franziska Bergemann, Lakshmy Vasan, Tamas Schauer, Anna Danese, Riccardo Bocchi, Maria Colomé-Tatché, Carol Schuurmans, Anna Philpott, Tobias Straub, Boyan Bonev, Magdalena Götz

Date Published: 2nd Jul 2024

Publication Type: Journal

Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Abstract (Expand)

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the … early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Authors: Lucas Caldi Gomes, Sonja Hänzelmann, Fabian Hausmann, Robin Khatri, Sergio Oller, Mojan Parvaz, Laura Tzeplaeff, Laura Pasetto, Marie Gebelin, Melanie Ebbing, Constantin Holzapfel, Stefano Fabrizio Columbro, Serena Scozzari, Johanna Knöferle, Isabell Cordts, Antonia F Demleitner, Marcus Deschauer, Claudia Dufke, Marc Sturm, Qihui Zhou, Pavol Zelina, Emma Sudria-Lopez, Tobias B Haack, Sebastian Streb, Magdalena Kuzma-Kozakiewicz, Dieter Edbauer, R Jeroen Pasterkamp, Endre Laczko, Hubert Rehrauer, Ralph Schlapbach, Christine Carapito, Valentina Bonetto, Stefan Bonn, Paul Lingor

Date Published: 1st Jul 2024

Publication Type: Journal

T cell-mediated microglial activation triggers myelin pathology in a mouse model of amyloidosis.

Abstract (Expand)

Age-related myelin damage induces inflammatory responses, yet its involvement in Alzheimer's disease remains uncertain, despite age being a major risk factor. Using a mouse model of Alzheimer's … disease, we found that amyloidosis itself triggers age-related oligodendrocyte and myelin damage. Mechanistically, CD8<sup>+</sup> T cells promote the progressive accumulation of abnormally interferon-activated microglia that display myelin-damaging activity. Thus, our data suggest that immune responses against myelinating oligodendrocytes may contribute to neurodegenerative diseases with amyloidosis.

Authors: Shreeya Kedia, Hao Ji, Ruoqing Feng, Peter Androvic, Lena Spieth, Lu Liu, Jonas Franz, Hanna Zdiarstek, Katrin Perez Anderson, Cem Kaboglu, Qian Liu, Nicola Mattugini, Fatma Cherif, Danilo Prtvar, Ludovico Cantuti-Castelvetri, Arthur Liesz, Martina Schifferer, Christine Stadelmann, Sabina Tahirovic, Özgün Gökçe, Mikael Simons

Date Published: 27th Jun 2024

Publication Type: Journal

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