Biology subjects

Henek, T.

Publications and source records attributed to Henek, T..

3 recordsLinked to original sources

bioRxiv2026

Open or closed? Exploring the conformational heterogeneity of human plasminogen across multiple resolution scales

Plasminogen (Plg), the zymogen of the fibrinolytic protease plasmin, is a multidomain, conformationally-rich protein that plays a crucial role in targeted thrombolysis for stroke and heart attack treatments. Yet, the conformational state that affects binding to Plg activators and fibrinolytic activity is rarely validated, contributing to poor reproducibility and failures in drug development. Here, we establish a structural biology workflow to characterize human Plg suspended in an aqueous solution, preserving it in a near-native state and eliminating the need for crystal growth. Two variants, cleavage-resistant Plg-RV (R561A) and catalytically inactive Plg-CAT (S741A), were expressed in mammalian cells. Both variants were correctly folded and stable (Tm {approx} 60.6{degrees}C) and, unlike commercial plasma-derived Plg, were resistant to staphylokinase-mediated activation. Small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry revealed substantial conformational heterogeneity. The recombinantly-produced Plg variants adopted a closed conformation, exhibiting a good fit to the closed structure determined by X-ray diffraction. Conversely, the plasma-derived Plg populated an extended, open-like state. Cryo-EM analysis of the Plg-RV variant yielded a 4.4 [A] resolution map, and a rigid-body-fitted model revealed the closed-state architecture. Our findings demonstrate that rigorous structural validation of Plg is essential for future functional studies and rational development of next-generation thrombolytic agents.

biochemistry↗

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↗