Biology subjects

Pusch, S.

Publications and source records attributed to Pusch, S..

5 recordsLinked to original sources

bioRxiv2026

Capturing early events in aryl hydrocarbon receptor activation using two complementary protein-protein interaction assays

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that enables cellular adaptation to environmental, nutritional and metabolic cues. Upon ligand binding, AHR translocates to the nucleus, heterodimerizes with the AHR nuclear translocator (ARNT) and regulates gene expression. Current approaches to measure AHR activity rely on transcriptional readouts, which vary depending on cell type and ligand. Here, we introduce two complementary protein-protein interaction-based assays that detect AHR activation by monitoring AHR-ARNT complex formation. Split-luciferase (NanoBiT) and bimolecular fluorescence complementation (BiFC) detect AHR activation independently of transcriptional output, capturing agonist- and antagonist-dependent AHR modulation across multiple ligands and cellular contexts. NanoBiT enables rapid, real-time analysis of AHR dimerization, whereas BiFC supports imaging of AHR interactions at subcellular resolution. The assays capture further attributes of AHR signaling, including dissociation from chaperones or HIF-1-mediated competition for ARNT, and enable detection of AHR activation in biological samples. Hence, both assays provide versatile tools to study AHR signaling.

biochemistry↗

bioRxiv2024

IDH-mutant gliomas arise from glial progenitor cells harboring the initial driver mutation

Discovering the cell-of-origin harboring the initial driver mutation provides a fundamental basis for understanding tumor evolution and development of new treatments. For isocitrate dehydrogenase (IDH)-mutant gliomas - the most common malignant primary brain tumors in adults under 50 - the cell-of-origin remains poorly understood. Here, using patient brain tissues and genome-edited mice, we identified glial progenitor cells (GPCs), including oligodendrocyte progenitor cells (OPCs), as the glioma-originating cell type harboring the IDH mutation as the initial driver mutation. We conducted comprehensive deep sequencing, including droplet digital PCR and deep panel and amplicon sequencing to 128 tissues from 62 patients (29 IDH-mutant gliomas and 33 IDH-negative controls) comprising tumors, normal cortex or normal subventricular zone (SVZ), and blood. Surprisingly, low-level IDH mutation was found in the normal cortex away from the tumor in 38.5% (10 of 26) of IDH-mutant glioma patients, whereas no IDH mutation was detected in the normal SVZ. Furthermore, by analyzing cell-type-specific mutations, the direction of clonal evolution, the single-cell transcriptome from patient brains and novel mouse model of IDH-mutant glioma arising from mutation-carrying OPCs, we determined that GPCs, including OPCs, harboring the initial driver mutation are responsible for the development and evolution of IDH-mutant gliomas. In summary, our results demonstrate that GPCs containing the IDH mutation are the cells-of-origin harboring the initial driver mutation in IDH-mutant gliomas.

cancer biology↗

bioRxiv2023

EMP3 sustains oncogenic EGFR/CDK2 signaling by restricting receptor degradation in glioblastoma

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7+ late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR-specific inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, this stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.

cancer biology↗

bioRxiv2022

Transkingdom mechanism of MAMP generation by chitotriosidase (CHIT1) feeds oligomeric chitin from fungal pathogens and allergens into TLR2-mediated innate immune sensing

Chitin is a highly abundant polysaccharide in nature and linked to immune recognition of fungal infections and asthma in humans. Ubiquitous in fungi and insects, chitin is absent in mammals and plants and, thus, represents a microbe-associated molecular pattern (MAMP). However, the highly polymeric chitin is insoluble, which potentially hampers recognition by host immune sensors. In plants, secreted chitinases degrade polymeric chitin into diffusible oligomers, which are fed to innate immune receptors and co-receptors. In human and murine immune cells, a similar enzymatic activity was shown for human chitotriosidase (CHIT1) and oligomeric chitin is sensed via an innate immune receptor, Toll-like receptor (TLR) 2. However, a complete system of generating MAMPs from chitin and feeding them into a specific receptor/co-receptor-aided sensing mechanism has remained unknown in mammals. Here, we show that the secreted chitinolytic host enzyme, CHIT1, converts inert polymeric chitin into diffusible oligomers that can be sensed by TLR1-TLR2 co-receptor/receptor heterodimers, a process promoted by the lipopolysaccharide binding protein (LBP) and CD14. Furthermore, we observed that Chit1 is induced via the {beta}-glucan receptor Dectin-1 upon direct contact of immortalized human macrophages to the fungal pathogen Candida albicans, whereas the defined fungal secreted aspartyl proteases, Sap2 and Sap6, from C. albicans were able to degrade CHIT1 in vitro. Our study shows the existence of an inducible system of MAMP generation in the human host that enables contact-independent immune activation by diffusible MAMP ligands with striking similarity to the plant kingdom. Moreover, this study highlights CHIT1 as a potential therapeutic target for TLR2-mediated inflammatory processes that are fueled by oligomeric chitin.

immunology↗

bioRxiv2020

G3BP1 tethers the TSC complex to lysosomes and suppresses mTORC1 in the absence of stress granules

G3BP1 (Ras GTPase-activating protein-binding protein 1) is widely recognized as a core component of stress granules (SG), non-membranous RNA-protein-assemblies required for cellular survival under stress. We report that in the absence of SG, G3BP1 acts as lysosomal anchor of the Tuberous Sclerosis Complex (TSC) protein complex. By tethering the TSC complex to lysosomes, G3BP1 suppresses signaling through the metabolic master regulator mTORC1 (mechanistic target of rapamycin complex 1). Like the known TSC complex subunits, G3BP1 suppresses phenotypes related to mTORC1 hyperactivity in the context of tumors and neuronal dysfunction. Thus, G3BP1 is not only a core component of SG but also a key element of lysosomal TSC-mTORC1 signaling. HighlightsThe bona fide stress granule component G3BP1 O_LIis a key element of the TSC-mTORC1 signaling axis. C_LIO_LItethers the TSC complex to lysosomes. C_LIO_LIprevents mTORC1 hyperactivation by metabolic stimuli. C_LIO_LIsuppresses mTORC1-driven cancer cell motility and epileptiform activity. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/044081v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@77cb4forg.highwire.dtl.DTLVardef@c4d1a0org.highwire.dtl.DTLVardef@19e83acorg.highwire.dtl.DTLVardef@1d6a378_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗