Biology subjects

Fritz, G.

Publications and source records attributed to Fritz, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Lef1 Promotes Foxi1-Dependent Ionocyte Specification by Airway Submucosal Gland Progenitors

Pulmonary ionocytes are rare cells in submucosal gland (SMG)-containing airways thought to have specialized ion transport properties that regulate airway surface fluid and mucus composition. The origins of pulmonary ionocytes remain unclear, but their association with airway SMGs suggests they may arise from specialized progenitors in this compartment. We show that mouse glandular myoepithelial cells (GMECs) have a unique capacity to specify ionocytes enriched in the Cftr channel. The Lef1 transcription factor was both necessary and sufficient to promote GMEC specification of ionocytes in mouse trachea, 3D organoids, and/or air-liquid interface (ALI) cultures. Overexpression of Lef1 (Lef1OE) in GMECs produced ionocyte-enriched epithelia with enhanced Cftr-mediated chloride, bicarbonate, and fluid transport properties. Moreover, Foxi1 knockout in Lef1OE GMECs prevented ionocyte formation while promoting specification of Cftr-expressing secretory cells. These findings provide a progenitor relationship for the anatomical association of ionocytes with SMGs and a transcriptional framework for coordinated specification of CFTR-expressing ionocytes and secretory cells.

cell biology↗

bioRxiv2026

Ncf1/Igfbp2 Signaling Regulates Airway Submucosal Gland Progenitor Fitness and Ionocyte Specification

Background: Airway submucosal gland progenitors (SMGPs) serve as reserve stem cells for epithelial regeneration, yet mechanisms governing their long-term maintenance remain poorly understood. Here, we identify Ncf1/Igfbp2 signaling as a regulator of mouse SMGP fitness and epithelial lineage specification. Methods: Mouse SMGPs with genetic disruption of Ncf1 or Igfbp2 were serially passaged to assess progenitor proliferation, organoid-forming capacity, epithelial marker expression, DNA damage, DNA repair responses, and telomere length. Differentiated epithelia were evaluated for Cftr-mediated anion transport and pulmonary ionocyte specification in vitro and in vivo. Results: Genetic disruption of Ncf1 or Igfbp2 preserved SMGP identity, enhanced proliferative and organoid-forming capacity, and reduced DNA damage accumulation through activation of DNA repair programs. Epithelia derived from Ncf1-/- and Igfbp2-/- SMGPs maintained higher Cftr-mediated anion transport at late passage and exhibited enhanced pulmonary ionocyte specification in vitro and in vivo. These effects occurred independently of telomere shortening, implicating altered stress responses and DNA damage handling rather than replicative limits. Conclusions: Together, our findings identify Ncf1/Igfbp2 signaling as a regulator of airway stem cell fitness and epithelial maturation, with implications for regenerative therapies in lung diseases such as cystic fibrosis.

cell biology↗

bioRxiv2026

Comparative analyses of prolonged stress responses of breast carcinoma cells after hypofractionated irradiation in vitro and in vivo

The anticancer efficacy of radiotherapy (RT) is limited by acquired radioresistance (RR). Here, we aim to characterize prolonged responses of breast carcinoma cells to hypofractionated irradiation (hFI). To this end, murine mammary 4T1 tumor cells (4T1WT) were subjected to a clinically oriented hFI protocol (56 Gy cumulative dose) to select radioresistant cells in vitro (4T1RS). Furthermore, hFI of subcutaneously growing 4T1CTR tumors (hFI; 24 Gy cumulative dose) was performed to radioselected 4T1IR cells in vivo. Following single irradiation in vitro, radioselected 4T1RS cells revealed increased proliferation, attenuated G2/M arrest and reduced apoptosis as compared to parental 4T1WT cells. Moreover, 4T1RS cells showed increased expression of DNA-damage response (DDR)-related proteins (pKAP1, pCHK2, {gamma}H2AX) and improved DSB repair efficiency as demonstrated by nuclear {gamma}H2AX foci analyses. The mRNA expression of factors regulating cell cycle progression, DDR, apoptosis and oxidative stress was substantially different between both cell variants in vitro. Ten days after hFI of in vivo growing tumors, residual DNA damage and apoptosis were increased in the radioselected 4T1IR tumors, whereas proliferation was reduced as compared to non-irradiated 4T1CTR control tumors. Both irradiated and non-irradiated tumors revealed complex differences in the mRNA expression profile of susceptibility- and metastasis related genes, including GADD45a, DUSP1, CDKN1a and NQO1 as well as CD44 and Rho-related factors, respectively. Moreover, hFI stimulated the infiltration of MPO-positive immune cells into tumor tissue while the presence of CD3-positive cells was reduced in the tumor area. In addition, hFI in vivo resulted in a dysregulated mRNA expression of various immune cell markers, Rho-regulatory factors, tissue remodeling molecules and cell adhesion factors. Summarizing, we identified long-lasting adaptive changes following hFI in vitro and in vivo that are associated with DNA replication, DNA repair, senescence and apoptosis as well as immune cell infiltration and tissue remodeling.

cell biology↗

bioRxiv2026

L-Lysine production from glucose and chitin monomers using engineered Vibrio natriegens

Despite its industrial importance, microbial L-lysine production has largely been confined to classical producer strains, leaving the fast-growing, non-pathogenic marine microorganism V. natriegens largely untapped as an unconventional biosynthetic platform. In this work, we established an L-lysine-overproducing V. natriegens DSM759 strain through a step-wise, systematic rational engineering strategy targeting the native biosynthetic pathway. Guided by our prior systems-level analysis of the strains genetic and regulatory architecture, we identified key metabolic bottlenecks and implemented knowledge-driven interventions to relieve pathway constraints. Central to production was alleviation of feedback inhibition in the native key regulatory enzymes, aspartate kinase (AK, lysC) and dihydrodipicolinate synthase (DHDPS, dapA). Site-directed amino-acid substitutions, replicating established E. coli feedback-resistance mechanisms, were introduced into conserved regions of the V. natriegens DSM759 enzymes, producing L-lysine-insensitive variants with kinetic parameters comparable to that of corresponding wild type enzymes. Among the tested configurations, the strain co-expressing Vn.lysC2 and Vn.dapA1:E84T reached the highest L-lysine titer (9.0{+/-}0.6 mM) and yield (0.11{+/-}0.01 molLys molGlc-1), whereas overexpression of additional L-lysine pathway genes provided no further benefit. Leveraging the hosts metabolic versatility, L-lysine synthesis was also demonstrated from the chitin-derived amino-sugar N-acetylglucosamine (0.09{+/-}0.00 molLys molGlcNAc-1), highlighting the potential to valorize chitin-rich waste streams from the seafood industry. This work establishes a minimal, rational strategy for L-lysine biosynthesis in V. natriegens DSM759 and positions it as a promising platform for sustainable amino acid production.

bioengineering↗

bioRxiv2026

The evolution of a Na+-sensitive Vibrio cholerae mutant unmasks the moonlighting aminopeptidase PepA as a regulator of nhaB Na+/H+ antiporter gene expression

The pathogenic bacterium Vibrio cholerae is native to seawater and can therefore be cultivated in nutrient media with increased salt concentration. To maintain osmotic balance, V. cholerae uses Na+/H+ antiporters and an Na+-translocating NADH:quinone oxidoreductase (Na+-NQR). The exact contribution of the various Na+/H+ antiporters to maintain Na+ and H+ homeostasis in V. cholerae is unclear. However, genetic studies indicate the major Na+/H+ antiporter NhaA and the Na+-NQR are required by the bacteria for Na+ resistance at alkaline conditions. Here we show that the growth defect of a V. cholerae nhaA nqr mutant at increased Na+ concentrations and alkaline pH is relieved by the rapid acquisition of suppressor mutations. The suppressor mutants could be assigned to two classes. We identified (i) mutations in the promoter of the nhaB gene encoding a Na+/H+ antiporter and (ii) mutations that either affect the expression level of the pepA gene or DNA binding activity of the encoded multifunctional aminopeptidase PepA. The characterization of the PnhaB and PpepA promoters of the suppressor mutants, membrane potential measurements and comparative proteome analyses identified PepA as a novel factor controlling Na+ homeostasis in V. cholerae. IMPORTANCEThe emergence and spread of multi-resistant bacteria are a major problem for humans and animals. Therefore, the identification of novel targets for the development of novel antibiotics to combat pathogenic bacteria is extremely important. Since sodium ion homeostasis is an essential process in many pathogenic bacteria, especially those living in marine environments, it represents an interesting target for antibiotics. The perturbation of sodium ion homeostasis does indeed impair the fitness of the human pathogenic bacterium Vibrio cholerae but can be restored by the rapid evolution of the bacteria. This suggests that inhibiting multiple targets with different antibiotics might be more effective in preventing the development of resistant bacteria.

microbiology↗

bioRxiv2025

Mouse precision-cut liver and kidney slices: an optimized ex vivo model for acute toxicity testing

As excretion organs, the kidneys and liver are exposed to high concentrations of potentially toxic substances. While animal models remain the gold standard for organ-specific toxicity testing, alternative ex vivo approaches are essential to align with the 3R principles (refinement, reduction, replacement). Precision-cut tissue slices (PCTS) retain native tissue architecture, cellular heterogeneity, the interplay of different cell types, and metabolic capacity, offering a promising link between in vitro and in vivo models. Here, we aimed to establish an optimized protocol for preparing and culturing precision-cut kidney and liver slices (PCKS and PCLS) from mice for use in substance-oriented toxicological tests. Key parameters - including slice thickness, media composition, oxygenation, glucose levels, and incubation time - were refined to maintain tissue viability and metabolic function. Five known toxins - acetaminophen, cyclosporin A, cisplatin, arsenic trioxide, and aristolochic acid I - were tested. While PCKS showed comparable sensitivity to established kidney cell lines, PCLS achieved IC50 values closely matching in vivo toxicity data. High reproducibility across different experimenters was achieved, highlighting the robustness of the model. In conclusion, this ex vivo system provides a valuable, reproducible, and ethically approved platform for acute nephrotoxicity and hepatotoxicity testing, supporting preclinical drug screening and potentially reducing reliance on animal experiments.

pharmacology and toxicology↗

bioRxiv2025

Mx1-Cre-mediated Rac1 knockout confers multiple protective effects against anthracycline-induced acute normal tissue injury

Pharmacological data point to RAC1 as promising target for protection against anthracycline-induced cardiotoxicity, yet supporting genetic evidence is limited. Moreover, the relevance of RAC1 for cross-organ injury and cross-agent-induced normal tissue toxicity is unknown. Here, we employed a Mx1-Cre-based mouse model that enables an inducible Rac1 knockout across different organs in order to comparatively analyze the influence of RAC1 on doxorubicin (DOX) and cisplatin (CisPt)-induced acute stress responses of the heart, kidney and liver. Following DOX treatment, the extend of DNA-double strand break (DSB) formation and the percentage of apoptotic cells were reduced in all three organs (i.e. heart, liver, kidney) of RAC1 deficient (Rac1-/-) animals as compared to the wildtype control (Rac1+/+). By contrast, in the absence of RAC1, CisPt-induced DNA damage was reduced only in the liver and the frequency of CisPt-stimulated apoptotic cell death remained unaffected by the Rac1 status in all organs. These findings demonstrate a strikingly organ- and agent-specific relevance of RAC1-regulated mechanisms for normal tissue damage evoked by genotoxic anticancer therapeutics. Accordingly, protein levels of phosphorylated DNA damage response (DDR)-related factors were also reduced in the kidney and liver of DOX treated Rac1-/- animals, but not in the heart. Yet, DOX-triggered mRNA expression of surrogate markers related to inflammation, fibrosis and senescence was preferentially reduced in the heart and kidney of Rac1 deficient mice. Taken together, RAC1 plays a so far unknown distinct role in the pathophysiology of anticancer drug-induced normal tissue damage by influencing DNA damage formation, activation of DDR, apoptosis induction as well as inflammation-, fibrosis- and senescence-associated responses in a pronounced agent- and organ-specific manner. Targeting of RAC1 appears particularly effective in the context of anthracycline-based therapeutic regimen for conferring broad organoprotection to both the heart and detoxifying organs. Translational PerspectiveThis study provides the first genetic evidence identifying RAC1 as a critical mediator of anthracycline-induced cardiac, hepatic and renal injury. We demonstrate that genetic deletion of Rac1 alleviates chemotherapy-induced DNA damage, apoptosis, and stress responses related to senescence, fibrosis, and inflammation in an agent- and organ-specific manner. These findings highlight RAC1 as a putatively clinically relevant target for the development of organoprotective pharmacological strategies aiming to improve the tolerability of anthracycline- and cisplatin-based therapeutic regimen and, accordingly, enhancing the quality of life and prognosis of tumor patients.

pharmacology and toxicology↗

bioRxiv2025

Establishing Vibrio natriegens as a high-performance host for acetate-based poly-3-hydroxybutyrate production

Acetate can be a sustainable and renewable carbon source which holds significant promise for biotechnological production but is underutilized industrially due to limited microbial efficiency. Vibrio natriegens, recognized for exceptionally fast growth rates, represents a compelling host for developing efficient acetate-based bioprocesses. In this study, adaptive laboratory evolution significantly enhanced V. natriegens ability to grow on acetate as the sole carbon source, achieving an 89% increase in growth rate. Genetic and transcriptomic analyses revealed key adaptations improving acetate uptake and metabolism via increased salt tolerance, boosted Pta/AckA pathway activity, and rewired quorum sensing. Further metabolic engineering and bioprocess optimization enabled the evolved strain to reach high cell densities and efficiently convert acetate into the bioplastic poly-3-hydroxybutyrate (PHB), with productivities up to 0.27 g/L/h and PHB accumulation reaching 45.66% of cell biomass. These advances position V. natriegens as a highly promising microbial platform for sustainable, scalable, and cost-effective biomanufacturing using acetate as green feedstock. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/661028v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@ebd5b0org.highwire.dtl.DTLVardef@11a28bforg.highwire.dtl.DTLVardef@6950aforg.highwire.dtl.DTLVardef@303d7b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Differential responses of murine embryonic stem cells (mESC) and their endothelial progeny to doxorubicin and pharmacological inhibitors of DNA repair and DNA damage response

The clinical use of the anticancer drug doxorubicin (Dox) is limited by irreversible cardiotoxicity. The detailed molecular mechanisms involved and the pathophysiological relevance of different cardiac cell types, including progenitor cells, are still unclear. Here, we investigated stress responses of murine embryonic stem cells (mESC), endothelial progenitor cells (EC d4) and terminally differentiated endothelial-like cells (EC d6) following exposure to Dox and selected pharmacological inhibitors of DNA repair and DNA damage response (DDR) (RAD51i B02 and HDACi entinostat (EST)). We found that EC d4 exhibited a pronounced Dox hypersensitivity as compared to both mESC and EC d6, which was independent of drug transport. Analysis of EdU incorporation and replication fork progression following drug treatment revealed substantial agent-specific differences between mESC and the various differentiation stages. Furthermore, cellular susceptibility to drug-induced formation of DNA damage (i.e. DSB and SSB) also changes with ongoing differentiation and drug treatment, with mESC and EC d4 / EC d6 being particular prone to enhanced residual SSB and DSB levels, respectively. Dox treatment of EC d4 did not affect their differentiation into EC d6, but caused multiple functional impairments of the surviving EC d6 progeny, including defects in mitochondrial homeostasis, barrier function related to cell-cell adhesion factors ZO1 and VE-cadherin, response to cytokine stimulation as well as LDL uptake. To summarize, we show substantial differences in the response of mESC, EC d4 and EC d6 to Dox and pharmacological inhibitors of DNA repair and DDR. Most important, treatment of EC d4 results in pronounced persisting functional impairments of differentiated EC d6, pointing to a transient particularly drug-sensitive time window during endothelial differentiation. These findings are important for hazard assessment in developmental toxicology and regenerative medicine in the context of anticancer drug-induced normal tissue damage. Highlights- EC d4 are most vulnerable towards Dox-induced cytotoxicity independent of drug transport - EC d4 and EC d6 display higher steady-state levels of drug-induced DSB as compared to mESC - Drug-induced replication stress is highest in mESC and decreases with differentiation. - Both Dox and DNA repair/DDR inhibitors damage mitochondrial homeostasis in EC d4 and EC d6 - Terminally differentiated EC d6 derived from drug-treated EC d4 display multiple functional impairments

pharmacology and toxicology↗

bioRxiv2025

SIRT1 limits neuronal fate following DNA damage

DNA damage is a major risk factor for the decline of neuronal functions with age and neurodegenerative diseases. The connection between DNA damage and neurodegeneration is extensively investigated, however, the mechanisms limiting the propagation of damaged DNA from highly replicative neural stem/progenitor cells (NSPCs) to post-mitotic neurons remains largely unknown. Here, we describe that enzymatic activity of the histone deacetylase sirtuin 1 (SIRT1) is important for the homologues recombination-dependent repair of double- stranded DNA breaks induced by etoposide. Furthemore, SIRT1 abolishes neuronal fate of murine NSPCs following induction of DNA damage. Pharmacological inhibition or genetic inactivation of SIRT1 rescues etoposide-mediated inhibition of neuronal differentiation in NSPCs and hippocampal slice cultures and promotes transcription of pro-neuronal genes. Inhibition of Ataxia-telangiectasia mutated (ATM), the central regulator of the DNA damage response, mimics the SIRT-dependent effect of DNA damage on neuronal differentiation, indicating that the ATM/SIRT1 axis inhibits formation of neuronal cells harbouring damaged DNA. These data are consistent with the role of SIRT1 in genome stability, healthy ageing and protection from the development of ageing-associated neurodegenerative diseases.

cell biology↗

bioRxiv2025

Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cell death

The therapeutic efficacy of anticancer therapeutics is limited by acquired drug resistance of tumor cells. Here, we aim to characterize and overcome resistance mechanisms of ovarian cancer cells to the anthracycline derivative doxorubicin (Doxo). To this end, comparative analyses of Doxo-induced stress responses of parental A2780 and Doxo-resistant A2780ADR variant were performed. A2780ADR cells revealed cross-resistance to multiple compounds, including anticancer drugs (cisplatin (CisPt), etoposide (Eto)) and DNA repair/ DNA damage response (DDR) inhibitors (olaparib, niraparib, entinostat, prexasertib, rabusertib). A2780ADR cells formed significantly less DNA double-strand breaks (DSB) after Doxo exposure as compared to A2780, resulting in a mitigated DDR, reduced proliferation inhibition and attenuated apoptosis. Potential resistance mechanisms identified to contribute to Doxo resistance of A2780ADR cells include increased Doxo efflux due to increased MDR1 expression and reduced topoisomerase II protein expression. Substantial resensitization of A2780ADR cells to Doxo was achieved by both the RAC1 GTPase inhibitor EHT1864, the histone deacetylase inhibitor entinostat (Est) and, most effectively, the calcium channel blocker verapamil (Ver). Notably, Ver-mediated sensitization also pertains to Eto and CisPt. The synergistic effect of Ver in combination with Doxo, which is reflected by low combination index (CI), likely involves inhibition of MDR1-mediated drug transport, leading to increased intracellular steady state levels of Doxo and elevated DNA damage formation, eventually promoting pro-apoptotic DDR. However, combination treatment with Doxo and Ver also increased the cytotoxic response of non-malignant murine cardiomyocytes (HL-1), murine embryonic stem cells (mESC) and human induced pluripotent stem cells (hiPSC). Based on our data we suggest inhibition of MDR1-mediated Doxo efflux by Ver as a useful approach to overcome acquired drug resistance of A2780ADR cells because it promotes DDR-related pro-death mechanisms, yet at the price of a potentially increased risk of normal tissue toxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/650268v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@43c0e5org.highwire.dtl.DTLVardef@2d1c5forg.highwire.dtl.DTLVardef@1a1d97borg.highwire.dtl.DTLVardef@cd4522_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Verapamil-mediated resensitization of Doxo-resistant tumor cells.As concluded from our data we suggest that verapamil increases the anticancer efficacy of doxorubicin (Doxo) in a synergistic manner in anticancer drug resistant ovarian A2780ADR cells. This is likely due to inhibition of MDR1-mediated drug export by Ver, leading to higher intracellular steady-state concentrations of Doxo. In consequence, Doxo-mediated Topo II poisoning is promoted, eventually causing increased DNA damage (DSB) formation and activation of DDR-related signaling mechanims, which in turn impair cell proliferation and stimulate cell death. Apart from verapamil, inhibition of Rac1 GTPase-regulated signaling by EHT1864 and inhibition of HDACs class I by the HDACi entinostat are also useful to overcome Doxo resistance of A2780ADR cells, yet with the exact molecular mechanisms involved being still unclear. C_FIG

pharmacology and toxicology↗

bioRxiv2025

Preconditioning of human iPSCs with doxorubicin causes genome-wide transcriptional reprogramming in iPSC-derived cardiomyocytes linked to mitochondrial dysfunction and impaired cardiac regeneration

BackgroundThe anthracycline doxorubicin (Dox) is a widely used genotoxic chemotherapeutic drug with known dose-limiting cardiotoxic effects. How Dox-induced damage either to cardiomyocytes or to cardiac stem cells, which may compromise cardiac regeneration, contributes to cardiotoxicity remains poorly understood. MethodsHere we used a human induced pluripotent stem cell (iPSC)-based model system to determine the sensitivity of stem cells (iPSCs) and iPSC-derived derived cardiomyocytes (iCMs) applying different treatment regimens of Dox. Next to a broad range of methods to determine cellular and mitochondrial functions we performed an in-depth whole genome transcriptome profiling in iPSCs as well as iCMs. ResultsAs compared to their differentiated counterparts, iPSCs are highly sensitive against even short pulse-treatments with low Dox concentrations. Using such rather mild treatment conditions, we observed major mitochondrial impairments as demonstrated by increased mitochondrial fragmentation, persistent loss of mitochondrial membrane potential, and reduced ATP levels, while neither a markedly increased nuclear DNA damage response nor apoptosis were detected. Albeit mitochondrial dysfunction was not accompanied by changes in mitochondrial ultrastructure or altered OXPHOS complex assembly, mitochondrial genome (mtDNA) organization was altered. This points to a possible role of mtDNA remodelling for contributing to the high susceptibility of iPSCs to Dox. Whole genome transcriptome profiling revealed major differences in the transcriptional response to Dox treatment between iPSCs and iCMs. We could show that a moderate and transient exposure of iPSCs to Dox is sufficient to cause major transcriptional changes as for example reflected by the downregulation of numerous pivotal genes regulating cellular homeostasis and energy metabolism in iPSCs. Furthermore, pulse-treatment with Dox at the iPSC stage, termed preconditioning here, shifts the global transcriptional landscape of iCMs towards the expression of genes associated with impaired cardiac muscle regeneration, disrupted energy metabolism, altered muscle contraction, and increased fibrosis. ConclusionsOur findings support the hypothesis that Dox-induced mitochondrial dysfunction and transcriptional preconditioning in stem cells results in an impaired regenerative capacity after differentiation. This highlights a potential critical role of stem cells in mediating Dox-induced cardiotoxicity.

cell biology↗

bioRxiv2025

High-Throughput Synthesis and Screening of a Cyanimide Library Identifies Selective Inhibitors of ISG15-specific Protease USP18

High-throughput screening (HTS) of (large) compound collections is a critical early step in many drug discovery programs, enabling the rapid identification of lead molecules with desirable biological activity. The success of HTS depends heavily on the quality of the compound libraries used, and as such, the development of targeted libraries has emerged as a promising approach to enhance the effectiveness of HTS efforts. However, the acquisition and synthesis of such libraries remain costly and labor-intensive, often yielding compound quantities far exceeding the small amounts required for screening. To address these challenges, we present a high-throughput synthesis-to-screening method for the efficient inplate generation and immediate HTS of a deubiquitinase (DUB)-focused compound library. Central to our approach is the use of an Echo acoustic liquid handler, which enables precise nanoliter-scale transfers of DMSO-based solutions, facilitating efficient and miniaturized synthesis directly in 1,536-well plates. Using this platform, we constructed a library of 7,536 compounds featuring a DUB-privileged cyanimide warhead and screened it against a panel of twelve DUBs and ubiquitin-like proteases. This identified two structurally related molecules with selective inhibitory activity against the interferon-stimulated gene 15 (ISG15) protease USP18, which we further developed into a first-in-class USP18 inhibitor with 35 nM potency. This compound, BB07CA902, demonstrated exceptional specificity for USP18 across a panel of 41 DUBs and effectively increased ISGylation levels in cells by inhibiting USP18 activity. With our technology, we enabled the efficient preparation of large DUB-targeted cyanimide-based libraries, which will accelerate future DUB inhibitor development.

biochemistry↗

bioRxiv2025

A NanoBRET-based assay monitoring interactions in the USP18 signaling hub identifies the first cell-penetrant small molecule compromising USP18/ISG15 binding

Protein modification by interferon-stimulated gene 15 (ISG15), termed ISGylation, exhibits antiviral properties and influences tumorigenesis, genome stability and metabolic processes. ISGylation is counteracted by the specific protease USP18. Likewise, viral proteases such as the papain-like protease (PLpro) from SARS-CoV-2 cleave ISG15 to undermine the host immune response. Beyond its role as a deISGylating enzyme, USP18 acts as a major negative regulator of the IFN signaling pathway in a STAT2-dependent manner. In humans, unconjugated ISG15 secures USP18 stability and the absence of USP18 or impaired STAT2/USP18 binding cause fatal interferonopathies. Thus, the USP18 signaling hub represents a critical checkpoint for type I IFN signaling and ISGylation, qualifying it as a promising immune and cancer drug target. However, suitable assays to monitor protein-protein interactions (PPIs) within the USP18/ISG15/STAT2 signaling hub and to screen for PPI modulators are missing and no specific inhibitors targeting USP18 interactions are available. To address this gap, we developed a method based on the NanoLuc luciferase (NLuc) Bioluminescence Energy Transfer (NanoBRET) assay system to study PPIs. Firstly, we generated stable cell lines suitable to monitor USP18/ISG15 and USP18/STAT2 interactions, providing a semi high-throughput screening (HTS)-compatible platform. In combination with a virtual pre-screen of 60,000 compounds against USP18 in silico, this assay allowed us to identify a first small molecule (ZHAWOC8655) that compromises cellular USP18/ISG15 binding and inhibits USP18 protease activity in vitro. To further explore the potential of using the NanoBRET system for testing PPI modulators, we evaluated the effect of GRL0617, a compound which was shown to disrupt the interaction between SARS-CoV-2 PLpro/ISG15 as well as SARS-CoV-2 PLpro/ubiquitin. NanoBRET based stable cell lines as presented here will be suitable for monitoring PPIs in other multiprotein complexes after various stimuli, mutations or small molecule administration and can be challenged with siRNA or CRISPR/Cas9 libraries to identify previously unrecognized regulators.

molecular biology↗

bioRxiv2024

USP24 is an ISG15 cross-reactive deubiquitinasethat mediates IFN-I production by de-ISGylatingthe RNA helicase MOV10

The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro. Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-{beta} stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-{beta} production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24s novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation.

biochemistry↗

bioRxiv2023

Perception of butenolides by Bacillus subtilis via the α/β-hydrolase RsbQ

The regulation of behavioural and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi1-3. DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous receptors that perceive strigolactones and karrikins, respectively, and that hydrolyse their ligands to effect signal transduction4-7. RsbQ, a homologue of D14 and KAI2 from the Gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB ({sigma}B)8,9. However, the molecular function of RsbQ is unknown. Here we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilised by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signalling function of RsbQ in vivo, with dGR24 being 10-fold more potent. Application of dGR24 to B. subtilis phenocopied loss-of-function rsbQ mutations and led to a significant down-regulation of {sigma}B-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signalling compounds between plants and bacteria to help shape rhizosphere communities.

microbiology↗

bioRxiv2023

USP16 is an ISG15 cross-reactive deubiquitinase targeting a subset of metabolic pathway-related proteins

The ubiquitin-like modifier ISG15 can modulate host and viral proteins to restrict viral and microbial infections, and act as a cytokine. Its expression and conjugation are strongly up-regulated by type I interferons. Here we identify the deubiquitinating enzyme USP16 as an ISG15 cross-reactive protease. Ubiquitin-specific protease 16 (USP16) was found to react with an ISG15 activity-based probe in pull-down experiments using chronic myeloid leukaemia-derived human cells (HAP1). Supporting this finding, recombinant USP16 cleaved pro-ISG15 and ISG15 iso-peptide linked model substrates in vitro, as well as ISGylated substrates present in cell lysates. Moreover, the interferon-induced stimulation of ISGylation in human HAP1 cells was increased by knockdown or knockout of USP16. Depletion of USP16 did not affect interferon signaling, and interferon treatment did not affect USP16 expression or enzymatic activity either. A USP16-dependent ISG15 interactome was established by anti-ISG15 immunoprecipitation mass spectrometry (IP-MS), which indicated that the deISGylating function of USP16 may regulate metabolic pathways involving GOT1, ALDOA, SOD1 and MDH1, all of which were further confirmed to be deISGylated by USP16 in HEK293T cells. Together, our results indicate that USP16 may contribute to regulating the ISGylation status of a subset of proteins related to metabolism during type I interferon responses.

biochemistry↗

bioRxiv2022

Novel regulatory logic in the antibiotic resistance response of Enterococcus faecalis against cell envelope targeting antibiotics

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how E. faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

microbiology↗