Biology subjects

Wahni, K.

Publications and source records attributed to Wahni, K..

5 recordsLinked to original sources

bioRxiv2026

Disease-linked mutations in hnRNPA2B1 accelerate condensate maturation and promote amyloid aggregation

Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is a multifunctional RNA-binding protein that undergoes liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles across different cellular contexts. However, dysregulated phase separation can drive the transition from functional condensates to pathological protein aggregates. Two mutations in the low-complexity domain (LCD) of hnRNPA2B1 (D302V and P310L) have been linked to neurodegenerative diseases. How these mutations alter the interplay between LLPS and aggregation remains poorly understood. Here, we show that these disease-linked mutations accelerate condensate maturation and promote amyloid-like aggregation of hnRNPA2B1, with fibrils emerging from condensate-like cores. To dissect the mechanistic link between phase separation, aggregation, and disease, we focused on a conserved 25-amino-acid region within the LCD harboring both mutation sites. Deletion of this region markedly impairs LLPS and abolishes aggregation, revealing its contribution to both processes. Isolated peptides derived from this region have an intrinsic propensity for amyloid-like fibril formation, which is enhanced by the mutations. Although unable to phase separate, these peptides remodel condensate morphology and nucleate aggregation of the LCD. Together, our results identify a sequence-encoded mechanism linking disease-associated mutations to altered condensate behavior and amyloid aggregation, providing molecular insights into how aberrant phase transitions may contribute to hnRNPA2B1-associated disease.

biochemistry↗

bioRxiv2026

Challenges in reconstituting the peroxiredoxin 2:STAT3 transient redox-relay complex in vitro

Peroxiredoxin 2 (Prdx2) mediates redox signaling by transferring oxidative equivalents to target proteins such as STAT3, a redox-sensitive transcription factor implicated in inflammation and cancer. Although this interaction has been demonstrated in cells, reconstituting the Prdx2:STAT3 complex in vitro remains challenging due to its transient and redox-dependent nature. Here we test various conditions to stabilize the complex between taggless Prdx2 and the core fragment of STAT3 (CF-STAT3), including oxidants, detergents, the facilitator Annexin A2, anaerobic environments, and CovalX crosslinking. Complex formation was assessed via mass photometry, analytical size-exclusion chromatography (SEC), SEC-MALS, and electron microscopy (EM). No stable complex was observed under standard conditions. Anaerobic environments briefly stabilized the interaction, but cryo-EM could not resolve the structure. CovalX crosslinking yielded short-lived but homogeneous complexes. We found that Prdx2 is highly susceptible to hyperoxidation at its peroxidatic cysteine, particularly in the presence of DTT or excess H2O2, resulting in loss of function. Maintaining non-reducing conditions during purification preserved Prdx2 in an oxidation-competent state, promoting formation of the disulfide bond between the peroxidatic and resolving cysteines and thereby enabling reproducible detection of a weak complex with CF-STAT3. Our findings establish a framework for studying redox-relay protein complexes in vitro and highlight the importance of oxidation state management during protein handling.

molecular biology↗

bioRxiv2025

Heterooligomerization drives structural plasticity of eukaryotic peroxiredoxins

Peroxiredoxins are highly conserved thiol peroxidases essential for peroxide detoxification, redox signaling, and chaperone activity. Prx1/AhpC-type peroxiredoxins are found throughout the eukaryotic kingdom, where multiple isoforms frequently coexist within the same cell and even in the same subcellular compartment. Long thought to form exclusively homooligomeric structures, we reveal that heterooligomerization is a conserved and important feature of eukaryotic Prx1/AhpC-type peroxiredoxins. We demonstrate that heterooligomer formation modulates peroxoredoxin oligomeric state and enhances structural stability. In yeast, Tsa1-Tsa2 peroxiredoxin heterodecamers form in response to oxidative stress and incorporated Tsa2 stabilizes the decameric state. Beyond yeast, we show that human PRDX1 and PRDX2, as well as plant and parasitic peroxiredoxins, engage in functional heterooligomerization. These findings challenge the long-held paradigm of peroxiredoxin homooligomerization and reveal a novel mechanism for regulating redox homeostasis. Our study provides new insights into peroxiredoxin structural plasticity with broad implications for redox biology, stress responses, and cellular adaptation.

biochemistry↗

bioRxiv2021

Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

Hypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

immunology↗

bioRxiv2020

Charge Interactions in a Highly Charge-depleted Protein

Interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions. SignificanceElectrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.

biochemistry↗