Biology subjects

Juhasz, T.

Publications and source records attributed to Juhasz, T..

3 recordsLinked to original sources

bioRxiv2025

Phase transition drives bacterial single-stranded DNA binding (SSB) protein mobilization during stress response and metabolic adaptation

Single-stranded DNA-binding proteins (SSBs) are ubiquitous factors of genome metabolism, recently recognized for their ability to undergo liquid-liquid phase separation (LLPS). While Escherichia coli SSB (EcSSB) has emerged as a model for bacterial LLPS in vitro, its phase behavior and functional dynamics in vivo have remained largely unexplored. Here, we define the subcellular organization of EcSSB under diverse physiological and stress conditions using super-resolution microscopy coupled with newly developed image analysis tools. We show that EcSSB forms dynamic intracellular assemblies during exponential growth, which partially dissolve in response to DNA damage, oxidative stress, antibiotic exposure, and metabolic adaptation. In contrast, these dynamic reorganizations are attenuated in stationary phase cells. Moreover, we show that EcSSB foci exhibit limited overlap with nucleoid regions under stress-free conditions, whereas stress induction is accompanied by increased DNA association. These in vivo observations are consistent with stress- and growth-phase dependent modulation of EcSSB organization and together with biophysical characterization of EcSSB condensation in vitro suggest a role for LLPS-based dynamic storage and mobilization in response to physiological demands. Our work provides a quantitative framework for analyzing the cellular organization of bacterial proteins and the spatial regulation of genome maintenance factors in changing environments. This knowledge may also support future strategies targeting SSB organization pathways for antimicrobial development.

biochemistry↗

bioRxiv2024

Residual flexibility in the topologically constrained multivalent complex between the GKAP scaffold and LC8 hub proteins

Guanylate kinase-associated protein (GKAP) is a large postsynaptic scaffold protein bearing two closely spaced noncanonical binding sites for the bivalent dynein light chain LC8 hub protein. This might allow the formation of heterogeneous complexes with different sizes and topologies. Here, we show that a well-defined hexameric complex is formed, composed of 2 GKAP molecules and 2 LC8 dimers. Using NMR spectroscopy, we demonstrate that the LC8-binding segment of GKAP is intrinsically disordered and the flexibility of the linker region is largely retained even in the complex form. Molecular dynamics calculations suggest that besides the tightly bound residues, the hexamer also exhibits several dynamically interchanging interactions. The flanking regions of the two binding sites on GKAP exhibit different interaction patterns, hinting at additional contacts that might explain the fixed stoichiometry of the assembly. Our results demonstrate that constrained stoichiometry can coexist with substantial flexibility in a multivalent system.

molecular biology↗

bioRxiv2023

Hypoxia-inducible factor (HIF) activation promotes osteogenic transition of valve interstitial cells and accelerates heart valve calcification in chronic kidney disease (CKD)

AimsValve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients on hemodialysis. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Methods and resultUsing in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1 and HIF-2) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1, HIF-2, Glut-1), and calcification in VICs. Down-regulation of HIF-1 and HIF-2 inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics (desferrioxamine, CoCl2, Daprodustat (DPD)) promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted VC in the CKD mice model. ConclusionsHIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1 and HIF-2, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate VC. Translational perspectiveOne in four hemodialysis-dependent CKD patients on DPD treatment experience a major cardiovascular event during a 2.5-year follow-up period. This work provides a possible explanation for this phenomenon and should initiate further studies to address whether DPD-mediated acceleration of valve calcification triggers the unbeneficial effect of DPD.

pathology↗