Biology subjects

Bauer, L. G.

Publications and source records attributed to Bauer, L. G..

4 recordsLinked to original sources

bioRxiv2025

Live-Cell Chemoproteomic Profiling Identifies the Uncharacterised Protein YbaA as a Direct Target of Ciprofloxacin in Escherichia coli

Fluoroquinolone antibiotics, such as ciprofloxacin, are important broad-spectrum agents for a range of bacterial infections; however, fluoroquinolone usage is increasingly challenged by the emergence of resistance. Ciprofloxacin resistance mechanisms include mutations in the antibiotic target DNA gyrase, downregulation of porins required for bacterial cell penetration, and upregulation of efflux pumps to expel the antibiotic. However, new pathways driving bacterial tolerance to fluoroquinolones are still being discovered, suggesting that additional ciprofloxacin-binding proteins may exist in bacteria. In this study, we report the use of affinity-based protein profiling (AfBPP) with photo-crosslinking chemical probes to identify protein binding partners of ciprofloxacin in live E. coli cells. AfBPP identified novel ciprofloxacin binding proteins including YjdN and YbaA, whose molecular functions are as yet unannotated. Target engagement was validated using genetic knockout and biophysical binding assays, and key interactions identified in the ciprofloxacin binding site of YbaA. Collectively, this study demonstrates that additional and previously unreported biological interactions can exist for well-established antibiotics, and provides methodology to identify and interrogate these interactions in detail.

microbiology↗

bioRxiv2025

Bisphosphonates Trigger Anti-Ageing Effects Across Multiple Cell Types and Protect Against Senescence

Bisphosphonates (BPs) have been the major class of medicines used to treat disorders of excessive bone loss for over five decades. Recently it has been recognized that BPs may also have additional significant beneficial extra-skeletal effects. These include a reduction of all-cause mortality and of conditions commonly linked to ageing, such as cancer and cardiovascular disease. Here we show that bisphosphonates co-localize with lysosomal and endosomal organelles in non-skeletal cells and stimulate cell growth at low doses. In vivo spatial transcriptomic analysis revealed differentially expressed senescence markers in multiple organs of aged BP-treated mice, and a shift in cellular composition toward those of young counterparts. Similarly, a 5000-plex plasma proteome analysis from osteopenic patients before and after BP-treatment showed significant alterations in [~]400 proteins including GTPase regulators and markers of senescence, autophagy, apoptosis, and inflammatory responses. Furthermore, treatment with BPs protected against the onset of senescence in vitro. Proteome-wide target deconvolution using 2D thermal profiling revealed novel BP-binding targets (PHB2, ASAH1), and combined with RNA- and ATAC-seq of BP-treated cells and patient data, suggests downstream regulation of the MEF2A transcription factor within the heart. Collectively, these results indicate how BPs may beneficially modify the human plasma proteome, and directly impact multiple non-skeletal cell types through previously unidentified proteins, thereby influencing a range of pathways related to senescence and ageing.

cell biology↗

bioRxiv2025

Targeted Protein Degradation of NUDT5 Reveals an Unexpected Non-Enzymatic Role in 6-Thioguanine-Mediated Toxicity

6-Thioguanine (6-TG), an FDA-approved antimetabolite drug, is widely used in the treatment of leukemia. Its cellular effects require metabolic activation and are regulated through interactions with various proteins such as NUDT15, which catalyzes the hydrolysis of the active 6-TG metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP. Recent genome-wide CRISPR loss-of-function studies have identified another NUDIX hydrolase, NUDT5, as a crucial mediator of 6-TG toxicity. Here, we present the development and characterization of potent and selective NUDT5 degraders, guided by a cell-based assay screening strategy. These degraders, in conjunction with orthogonal CRISPR knock-out and reconstitution experiments, reveal a novel and unexpected, non-enzymatic role for NUDT5 in modulating the cellular response to 6-TG. Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting a distinct mode-of-action with potential implications for patient therapy.

cell biology↗

bioRxiv2025

Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. Here, we investigate the chemical and genetic dependencies caused by mutations in the folate enzyme MTHFD1 and discover a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Through genetic knockout and development of a selective chemical NUDT5 degrader, we uncover an unprecedented scaffolding role rather than NUDT5 enzymatic activity is responsible for this phenotype. We find that NUDT5 interacts with the rate-limiting enzyme of purine de novo synthesis, PPAT, to repress the pathway in response to elevated purine levels. Our findings establish NUDT5 as an important regulator of purine de novo synthesis and elucidate its role in mediating sensitivities to 6-thioguanine in cancer treatment and to adenosine in MTHFD1 deficiency.

molecular biology↗