Biology subjects

Verhee, A.

Publications and source records attributed to Verhee, A..

6 recordsLinked to original sources

bioRxiv2025

Optimizing efficacy to safety ratio of glucocorticoids in rheumatoid arthritis models by leveraging PPARα agonism

ObjectivesGlucocorticoids remain essential therapies for several immune- and inflammatory diseases such as rheumatoid arthritis (RA) but are notorious for their (metabolic) side effects. Given the anti-inflammatory and metabolically favorable actions of peroxisome-proliferator activated nuclear receptor (PPAR) agonists, we investigated whether PPAR agonism could enhance the therapeutic efficacy and/or mitigate the (metabolic) side effects of glucocorticoids. MethodsWe evaluated the effects of the synthetic glucocorticoid dexamethasone and the PPAR agonist GW7647 (GW) across three RA model systems: L929sA fibroblasts, primary human fibroblast-like synoviocytes (FLS) and collagen-induced arthritis (CIA) mice. ResultsDexamethasone reduced the inflammatory TNF response in L929sA cells, which was further potentiated by GW. In vivo however, GW reduced the dexamethasone-induced adiposity and hypertriglyceridemia, but not arthritis severity. Curiously, GW alone induced several proinflammatory genes within arthritic synovium which were counteracted by glucocorticoids. Proteomic profiling of TNF-stimulated human FLS revealed that combined use of dexamethasone and GW selectively suppressed interferon-stimulated proteins. In line herewith, co-stimulation with TNF and IFN{beta} amplified the suppressive effect of combined dexamethasone and GW treatment on pro-inflammatory gene expression in L929sA versus TNF alone. ConclusionGW enhances the anti-inflammatory effects of glucocorticoids in human FLS and L929sA, and mitigates metabolic side effects of dexamethasone in vivo, without compromising their efficacy. In addition, PPAR agonism permits to broaden its anti-inflammatory profile to interferon driven pathways. Given that both synthetic glucocorticoids and PPAR agonists are already widely used in (general) clinical practice, these findings offer a promising strategy to optimize glucocorticoid-based therapies. KEY MESSAGESO_ST_ABSWhat is already known on this topicC_ST_ABSO_LISynthetic glucocorticoids such as dexamethasone are widely used as immunosuppressive drugs, but cause many (metabolic) side effects. C_LIO_LIPeroxisome-proliferator-activated nuclear receptor (PPAR) agonists are clinically primarily used to treat symptoms that resemble glucocorticoid-induced side effects, but they also exert (modest) immunosuppressive effects. C_LI What this study addsO_LIThis study explores the therapeutic potential of a combination treatment with dexamethasone and PPAR agonist GW7647 (GW) in cellular and murine models of rheumatoid arthritis C_LIO_LIWe reveal that GW attenuates dexamethasone-induced adiposity and hypertriglyceridemia in vivo, hereby improving glucocorticoid-related side effects. C_LIO_LIThe therapeutic efficacy of dexamethasone is maintained or even enhanced by GW in murine and cellular models of rheumatic arthritis, respectively. C_LIO_LIWe offer novel mechanistic insights in the proposed combination treatment by revealing the selective suppression of interferon signaling pathways in human FLS. C_LI How this study might affect research, practice or policyO_LIGiven that both synthetic glucocorticoids and PPAR agonists are already used in clinical practice, this study offers a promising, translatable strategy to optimize glucocorticoid-based therapies with an improved efficacy/safety ratio. C_LI

immunology↗

bioRxiv2025

Rapid assay optimization by plug-and-play MAPPIT

Studying protein-protein interactions, which are crucial for all cellular processes in both health and disease, offers valuable insights into underlying mechanisms and reveals promising therapeutic targets. While efforts have been made to vastly increase the scale of MAPPIT assays, covering the full protein interactome remains challenging, in particular when considering the availability of different assay configurations. In this study, we expanded our Golden Gate modular cloning platform to enable rapid, customizable construct assembly for MAPPIT assays. We demonstrate the strength of this expanded toolbox for multiple distinct protein-protein interactions, including a MAPPIT assay for six different intracellular VHH targeting the anti-viral effector protein MXA.

synthetic biology↗

bioRxiv2025

Virotrap Reveals Salmonella SopB as A Ubiquitinated Cargo for Host ESCRT-0

The pathogenic bacterium Salmonella survives and replicates in host cells within a Salmonella-containing vacuole (SCV), a membranous niche it actively remodels via secreted effectors. The phosphoinositide phosphatase SopB is a key effector implicated in SCV biogenesis, yet its host protein interactome has remained incompletely defined. Using the mass spectrometry-based interactomics technology Virotrap, we identify a novel set of SopB-associated host proteins, including the ESCRT-0 subunit HGS and other membrane remodeling proteins. We demonstrate that SopB directly interacts with HGS via its ubiquitin-binding domains, and show that SopB promotes ESCRT-0 recruitment to the SCV, yet ultimately counteracts its anti-replicative effect. As the ESCRT machinery plays a central role in cargo sorting and membrane remodeling, we propose a new SopB-dependent strategy by which Salmonella hijacks host endosomal trafficking to build its intracellular niche.

microbiology↗

bioRxiv2024

CCR1 inhibition sensitizes multiple myeloma cells toglucocorticoid therapy

Glucocorticoids (GC) are cornerstone drugs in the treatment of multiple myeloma (MM). Because MM cells exploit the bone marrow microenvironment to obtain growth and survival signals, resistance to glucocorticoid-induced apoptosis emerges, yet the underlying mechanisms remain poorly characterized. Here, we identify that the chemokine receptor CCR1, together with its main ligand CCL3, plays a pivotal role in reducing the glucocorticoid sensitivity of MM cells. We show that blocking CCR1 signaling with the antagonist BX471 enhances the anti-MM effects of the glucocorticoid dexamethasone in MM cell lines, primary patient material and a myeloma xenograft mouse model. Mechanistically, the drug combination shifts the balance between pro- and antiapoptotic proteins towards apoptosis and deregulates lysosomal proteins. Our findings suggest that CCR1 may play a role in glucocorticoid resistance, as the GC-induced downregulation of CCR1 mRNA and protein is blunted in a GC-resistance onset model. Moreover, we demonstrate that inhibiting CCR1 partially reverses this resistance, providing a promising strategy for resensitizing MM cells to GC treatment.

cancer biology↗

bioRxiv2023

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein-of-interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split-link design, we applied it to identify protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field. MotivationIn proximity labeling proteomics protein-protein interactions are identified by in vivo biotinylation. However, the current lack of a universally applicable negative control for differential analysis affects accurate mapping of the interactome. To bridge this gap, we conceptualized a system based on the T2A self-cleaving peptide to match expression levels between control and bait protein setups while using the same bait protein. In addition, we implemented a versatile modular cloning system to build mammalian expression vectors for, but not limited to, proximity labeling assays.

biochemistry↗

bioRxiv2023

Antagonism and selective modulation of the human glucocorticoid receptor both reduce recruitment of p300/CBP and the Mediator complex

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with agonists, antagonists, or lead selective GR agonists and modulators (SEGRAMs), we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that the GR antagonist RU486 and the SEGRAM Dagrocorat both reduced GR interaction with CREB-binding protein (CBP)/p300 and the Mediator complex when compared to the full GR agonist Dexamethasone. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses. In BriefGlucocorticoids are commonly prescribed for the treatment of inflammatory disorders but are associated with severe side effects. Novel glucocorticoid receptor (GR) ligands with strong anti-inflammatory effects but reduced side effects are still sought after. Despite decades-long GR research, there is still an incomplete understanding of the molecular mechanisms driving context-specific GR activity. Using proximity labeling proteomics, we identified CREB-binding protein (CBP), p300 and the Mediator complex as potential crucial GR coregulators driving ligand-induced changes in GRs transcriptional activity. HighlightsO_LIGlucocorticoids (GCs), potent anti-inflammatory agents, can elicit side effects C_LIO_LIMore selective GCs, causing less side effects, are currently still unavailable C_LIO_LILack of fundamental insights on context-specific actions of the GC receptor (GR) C_LIO_LIWe mapped ligand-specific GR interactomes using proximity labeling proteomics C_LIO_LIp300/CBP and Mediator undergo ligand-dependent changes in interaction with GR C_LI

molecular biology↗