Biology subjects

Kucera, J.

Publications and source records attributed to Kucera, J..

8 recordsLinked to original sources

bioRxiv2026

Investigating the Conformational Flexibility of Staphylokinase Across Multiple Time Scales

Cardiovascular diseases, including ischemic stroke, necessitate improved thrombolytic agents. A microbe-encoded plasminogen activator staphylokinase (SAK) is a promising alternative to the widely used tissue plasminogen activator (tPA) due to its high fibrin specificity and low production cost. To overcome potential immunogenicity hampering its use in clinical settings, the low-immunogenic variants SAK SY155 and SAK THR174 were previously engineered. However, the molecular basis underlying their reduced immunogenicity is not understood and requires detailed elucidation. Here, we determine molecular structures and compare flexibility between low-immunogenic and immunogenic SAK variants, using a combination of experimental and computational structural techniques. Our analyses show that all variants share the canonical SAK fold and retain similar plasminogen activation kinetics, despite the number of introduced substitutions. Crucially, the low-immunogenic variants exhibit distinct flexibility profiles, with SAK THR174 showing substantially increased flexibility in the H1 helix and B3 region. SAK SY155 exhibits an increased flexibility in the H1-B3 loop and propensity to homodimerize. These flexibility changes are found in the known immunogenic epitopes. Our multi-scale flexibility analysis provides the molecular explanation for the reduced immunogenicity, altered thermostability, and retained fibrinolytic function of the engineered variants. This information is critical for the design of next-generation thrombolytics.

biochemistry↗

bioRxiv2025

Taurine Inhibits Apolipoprotein E4 Aggregation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor in many neurodegenerative diseases, yet effective therapeutic strategies targeting its associated pathologies remain unresolved. The aggregation of ApoE4, a key pathological feature, can be attenuated by tramiprosate and its metabolite 3-sulfopropanoic acid. In this study, we investigated the potential of taurine, a close chemical analogue of tramiprosate, to modulate ApoE4-mediated pathological processes. Using an integrated approach--including molecular dynamics simulations, static light scattering, mass spectrometry, and cerebral organoid models--we investigated taurines effects on ApoE4 aggregation. We found that taurine effectively inhibits ApoE4 aggregation. Notably, taurine significantly ameliorates the pathophysiological characteristics of ApoE4, bringing its phenotype closer to the more benign ApoE3 variant. By leveraging its neuroprotective properties, taurine may offer effects comparable to tramiprosate and 3-sulfopropanoic acid, positioning it as an accessible and promising candidate for mitigating neurodegeneration, particularly in individuals with the high-risk ApoE4/E4 genotype.

biochemistry↗

bioRxiv2025

All-optical Diamond Heater-Thermometer enables versatile and reliable thermal modulation of ion channels at the single-cell level

A living cell is a nonequilibrium thermodynamic system where, nevertheless, a notion of local equilibria exists. This notion applies to all micro- and nanoscale aqueous volumes, each containing a large number of molecules. This allows one to define sets of local conditions, including thermodynamic ones; for instance, a defined temperature requires thermodynamic equilibrium by definition. Once such a condition is fulfilled, one can control local variables and their gradients to theoretically describe the thermodynamic state of living systems at the micro- and nanoscale. Performing ultralocal experimental manipulations has become possible thanks to the patch clamp technique to control the cell membrane potential, as well as fluorescent imaging to monitor molecular concentrations and their intracellular gradients. However, precise temperature gradient control at the micro-/nanoscale has yet lacked a reliable experimental realization in a living cell. Here, we present a new methodology - microscale control of a temperature gradient profile in aqueous media by a fully optical Diamond Heater-Thermometer in a plug-and-play configuration combined with the patch clamp technique. In particular, we demonstrate applications of the combined Diamond Heater-Thermometer-patch clamp approach for the fast and reproducible thermal modulation of ionic current from voltage-gated Nav1.5 sodium channels expressed in HEK293 cells and freshly isolated ventricular mouse cardiomyocytes. Such an approach of manipulating the ultra-local temperature down to the nanoscale has the potential to uncover previously inaccessible phenomena in various physiological intracellular processes related to the endogenous nanoscale heat sources, such as open ion channels capable of producing Joule heat.

biophysics↗

bioRxiv2021

Biological Factors and Self-Perception of Stress Predict Human Freeze-Like Responses in the Context of Self-Defence Training and Personal Experience with Violence

Many animals react to threatening stimuli such as a predator attacks by freezing. However, little experimental research investigated freeze response in humans. Here, we have employed practices commonly used in self-defence training to create two unique scenarios simulating armed physical attacks. Sixty healthy men volunteers divided into three groups of twenty (untrained, trained but unexperienced, trained and experienced) underwent these scenarios accompanied by measurement of biochemical, physiological, and psychological markers of stress. All participants also underwent an fMRI session during which they observed neutral and negative images from the International Affective Picture System (IAPS). Our results show that scenarios simulating physical attacks can induce a freeze-like response in men. In addition, we demonstrate that while electrodermal activity (EDA), subjective stress perception, and brain activity in fMRI predict freeze-like response in men, their effect on freeze-like response is entirely dependent on the level of training and experience of a given individual.

neuroscience↗

bioRxiv2021

Kinetic analysis of ASIC1a delineates conformational signaling from proton sensing domains to the channel gate

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. Molecular dynamics simulations were used to further evaluate VCF-predicted movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

neuroscience↗

bioRxiv2020

Cytotoxicity of the effector protein BteA was attenuated in Bordetella pertussis by insertion of an alanine residue

Bordetella bronchiseptica and Bordetella pertussis are closely related respiratory pathogens that evolved from a common bacterial ancestor. While B. bronchiseptica has an environmental reservoir and mostly establishes chronic infections in a broad range of mammals, B. pertussis is a human-specific pathogen causing acute pulmonary pertussis in infants and whooping cough illness in older humans. Both species employ a type III secretion system (T3SS) to inject a cytotoxic BteA effector protein into host cells. However, compared to the high BteA-mediated cytotoxicity of B. bronchiseptica, the cytotoxicity induced by B. pertussis BteA (Bp BteA) appears to be quite low and this has been attributed to the reduced T3SS gene expression in B. pertussis. We show that presence of an alanine residue inserted at position 503 (A503) of Bp BteA accounts for its strongly attenuated cytotoxic potency. Deletion of A503 from Bp BteA greatly enhanced the cytotoxic activity of B. pertussis B1917 on mammalian HeLa cells and expression of Bp BteA{Delta}A503 was highly toxic to Saccharomyces cerevisiae cells. Vice versa, insertion of A503 into B. bronchiseptica BteA (Bb BteA) strongly decreased its cytotoxicity to yeast and HeLa cells. Moreover, production of Bp BteA{Delta}A503 increased virulence of B. pertussis B1917 in the mouse model of intranasal infection (reduced LD50) but yielded less inflammatory pathology in infected mouse lungs at sublethal infectious doses. This suggests that A503 insertion in the T3SS effector Bp BteA may represent an evolutionary adaptation that fine-tunes B. pertussis virulence and host immune response. Author summaryPertussis remains the least-controlled vaccine-preventable infectious disease and the mechanisms by which Bordetella pertussis subverts defense mechanisms of human airway mucosa remain poorly understood. We found that B. pertussis had the cytotoxic activity of its type III secretion system-delivered effector BteA strongly attenuated by insertion of an alanine residue at position 503 as compared to the BteA homologue of the animal pathogen B. bronchiseptica. This functional adaptation reduced the capacity of B. pertussis to suppress host inflammatory response and may contribute to an acute course of the pulmonary form of human infant pertussis.

microbiology↗

bioRxiv2020

FliI6-FliJ molecular motor assists with unfolding in the type III secretion export apparatus

The role of rotational molecular motors of the ATP synthase class is integral to the metabolism of cells. Yet the function of FliI6-FliJ complex - a homolog of the F1 ATPase motor - within the flagellar export apparatus remains unclear. We use a simple two-state model adapted from studies of linear molecular motors to identify key features of this motor. The two states are the locked ground state where the FliJ coiled coil filament experiences fluctuations in an asymmetric torsional potential, and a free excited state in which FliJ undergoes rotational diffusion. Michaelis-Menten kinetics was used to treat transitions between these two states, and obtain the average angular velocity of the FliJ filament within the FliI6 stator:{omega} max {approx} 9.0 rps. The motor was then studied under external counter torque conditions in order to ascertain its maximal power output: Pmax {approx} 42 kBT/s, and the stall torque: Gstall {approx} 3 kBT/rad. Two modes of action within the flagellar export apparatus are proposed, in which the motor performs useful work either by continuously grinding through the resistive environment, or by exerting equal and opposite stall force on it. In both cases, the resistance is provided by flagellin subunits entering the flagellar export channel prior to their unfolding. We therefore propose that the function of the FliI6-FliJ complex is to lower the energy barrier and therefore assist in unfolding of the flagellar proteins before feeding them into the transport channel.

biophysics↗

bioRxiv2017

Peripatric speciation associated with genome expansion and female-biased sex ratios in the moss genus Ceratodon

PREMISE OF THE STUDYA period of allopatry is widely believed to be essential for the evolution of reproductive isolation. However, strict allopatry may be difficult to achieve in some cosmopolitan, spore-dispersed groups, like mosses. Here we examine the genetic and genome size diversity in Mediterranean populations of the moss Ceratodon purpureus s.l. to evaluate the role of allopatry and ploidy change in population divergence.\n\nMETHODSWe sampled populations of the genus Ceratodon from mountainous areas and lowlands of the Mediterranean region, and from western and central Europe. We performed phylogenetic and coalescent analyses on sequences from five nuclear introns and a chloroplast locus to reconstruct their evolutionary history. We also estimated the genome size using flow cytometry, employing propidium iodide, and determined their sex using a sex-linked PCR marker.\n\nKEY RESULTSTwo well differentiated clades were resolved, discriminating two homogeneous groups: the widespread C. purpureus and a local group mostly restricted to the mountains in southern Spain. The latter also possessed a genome size 25% larger than the widespread C. purpureus, and the samples of this group consist entirely of females. We also found hybrids, and some of them had a genome size equivalent to the sum of the C. purpureus and Spanish genome, suggesting that they arose by allopolyploidy.\n\nCONCLUSIONSThese data suggest that a new species of Ceratodon arose via peripatric speciation, potentially involving a genome size change and a strong female-biased sex ratio. The new species has hybridized in the past with C. purpureus.

molecular biology↗