Biology subjects

Fiala, J.

Publications and source records attributed to Fiala, J..

4 recordsLinked to original sources

bioRxiv2025

Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 [A] structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

molecular biology↗

bioRxiv2023

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs

Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-targets, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy, PhosID-ABPP, on a timsTOF HT mass spectrometer. This refinement enables dual dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely PF-06672131 and PF-6422899, facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, while both ABPs showed comparable affinities for the EGFR, PF-06672131 had a broader off-target reactivity profile. In contrast, PF-6422899 exhibited higher affinity for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF-06672131 also effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nanomolar. Additionally, analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

biochemistry↗

bioRxiv2021

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. Oxidative modifications were precisely localized on specific residues using a timsTOF Pro mass spectrometer. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Data are available via ProteomeXchange with identifier PXD021621.

biochemistry↗

bioRxiv2021

A new High-Throughput-Screening-assay for Photoantimicrobials Based on EUCAST Revealed Unknown Photoantimicrobials in Cortinariaceae

A spark of light might unravel antimicrobial activity of colored compounds, which otherwise would have been classified as inactive. While many mushrooms contain such colorful pigments but lack antimicrobial activity, we wondered if a controlled irradiation is needed to unleash their effect. To explore such photoantimicrobial actions in the Kingdom Fungi, an efficient high-throughput-screening (HTS) assay is needed. Here we report on the establishment of a reliable photoantimicrobial assay based on the EUCAST recommendations, which was validated with known photosensitzers (i.e., curcumin, phenalenone, rose bengal, an hypericum extract, and methylene blue). Furthermore, an improved LED-irradiation setup enabling with only 24 LEDs a homogenous irradiation of a 96-well plate is presented. The established HTS-assay was utilized to screen six colorful Cortinarius extracts unrevealing C. xanthophyllus and C. rufo-olivaceus as promising sources for new photoantimicrobials.

microbiology↗