Biology subjects

Kozak, M.

Publications and source records attributed to Kozak, M..

7 recordsLinked to original sources

bioRxiv2025

Inducible tomato defences persist in detached leaves, despite differential plant variety and gene-dependent expression

Herbivore and pathogen attacks often lead to the induction of plant defences. Given the intricate nature of such responses, plant integrity is assumed to be a pre-requisite for the successful mounting of defences, a hypothesis supported by work done in plant-pathogen interactions. However, the relevance of plant integrity in plant-herbivore interactions, particularly in direct plant defences, is unclear and lacks empirical molecular validation. To test this, we measured the expression of plant defence-related genes (PPO-D and PI-IIc, involved in the jasmonate signal pathway and PR-1a, involved in the salicylic acid signal pathway) in intact plants and detached leaves of two tomato varieties infested or not with the herbivorous spider mite Tetranychus urticae. We also tested whether defences persisted in detached leaves after one, four, seven or fourteen days of infestation. We found that tomato defences were induced in both intact plants and detached leaves and that the expression of all three genes in detached leaves increased over time, being the increment higher in the two late-expression genes, PI-IIc and PR-1a. Such results suggests that both intact plants and detached leaves can be used in studies addressing herbivore interactions with plant defences. However, induction levels in intact plants and detached leaves varied with plant variety and the gene assessed, ranging from higher expression in detached leaves than in intact plants to the reverse. Hence, studies aiming at identifying the role of specific genes should account for the differential, variety-dependent, role of plant integrity in their expression.

ecology↗

bioRxiv2023

Characterization of uranyl ion binding to amyloid beta (Aβ) peptides: effects on Aβ structure and aggregation

AbstractUranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions including Alzheimers disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is deposition of amyloid plaques, consisting mainly of amyloid-{beta} (A{beta}) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric A{beta} aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase A{beta} production, and these metals bind to A{beta} peptides and modulate their aggregation. Possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between A{beta} peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to A{beta} peptides with affinities in the micromolar range, induce structural changes in A{beta} monomers and oligomers, and inhibit A{beta} fibrillization. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation.

biophysics↗

bioRxiv2022

Lactose-based surfactants complexable with oligonucleic acids as gene delivery agents

Advances in molecular medicine related to the treatment of genetic disorders and cancer require finding new pathways for gene transfer. Nonviral delivery methods rely on the application of polymers, liposomes and cationic lipid systems used as vehicle. Among these systems, there is increasing interest in surfactants, which, due to their ability to complex with nucleic acids can deliver into cell DNA or RNA molecules of almost any size, which is unattainable with viral gene delivery systems.The main aim of this study was to determine the effect of the concentration of lactose-based surfactants (zwitterionic derivatives of sulfobetaine with carbohydrate moieties) on the structure of DNA/RNA as well as to explore their abilities of nucleic acid complexation. Structural studies of DNA or RNA in complexes with surfactants of two types at various concentrations were conducted using circular dichroism (CD) spectroscopy, gel electrophoresis (GE) and synchrotron radiation small angle X-ray scattering (SR-SAXS). Our studies showed that the examined surfactants have excellent properties of forming complexes with DNA and RNA. Additionally, to determine the cytotoxicity and transfection abilities of the studied lipoplexes, preliminary tests were performed in HeLa and fibroblast cells. The obtained results suggest that these systems have relatively low toxicity; however, further research is needed in this area.

biophysics↗

bioRxiv2021

The Prion Protein Octarepeat Domain Forms Transient β-sheet Structures Upon Residue-Specific Cu(II) and Zn(II) Binding

Misfolding of the cellular prion protein (PrPC) is associated with the development of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). Metal ions appear to play a crucial role in the protein misfolding, and metal imbalance may be part of TSE pathologies. PrPC is a combined Cu(II) and Zn(II) metal binding protein, where the main metal binding site is located in the octarepeat (OR) region. Here, we used biophysical methods to characterize Cu(II) and Zn(II) binding to the isolated OR region. Circular dichroism (CD) spectroscopy data suggest that the OR domain binds up to four Cu(II) ions or two Zn(II) ions. Upon metal binding, the OR region seems to adopt a transient antiparallel {beta}-sheet hairpin structure. Fluorescence spectroscopy data indicates that under neutral conditions, the OR region can bind both Cu(II) and Zn(II) ions, whereas under acidic conditions it binds only Cu(II) ions. Molecular dynamics simulations suggest that binding of both metal ions to the OR region results in formation of {beta}-hairpin structures. As formation of {beta}-sheet structures is a first step towards amyloid formation, we propose that high concentrations of either Cu(II) or Zn(II) ions may have a pro-amyloid effect in TSEs.

biophysics↗

bioRxiv2021

The engineered peptide construct NCAM1-Aβ inhibits aggregation of the human prion protein (PrP)

In prion diseases, the prion protein (PrP) becomes misfolded and forms fibrillar aggregates, which are resistant to proteinase degradation and become responsible for prion infectivity and pathology. So far, no drug or treatment procedures have been approved for prion disease treatment. We have previously shown that engineered cell-penetrating peptide constructs can reduce the amount of prion aggregates in infected cells. The molecular mechanisms underlying this effect are however unknown. Here, we use atomic force microscopy (AFM) imaging to show that the aggregation of the human PrP protein can be inhibited by equimolar amounts of the 25 residues long engineered peptide construct NCAM1-A{beta}.

biochemistry↗

bioRxiv2019

Phase separation of Ede1 promotes the initiation of endocytic events

Clathrin-mediated endocytosis is a major pathway that eukaryotic cells use to produce transport vesicles from the plasma membrane. The assembly of the endocytic coat is initiated by a dynamic network of weakly interacting proteins, but the exact mechanism of initiation is unknown. Ede1, the yeast homologue of mammalian Eps15, is one of the early-arriving endocytic proteins and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and the frequency of endocytic initiation is decreased. We show here that in mutants with increased amounts of cytoplasmic Ede1, the excess protein forms large condensates which exhibit properties of phase separated liquid protein droplets. These Ede1 condensates recruit many other early-arriving endocytic proteins. Their formation depends on the core region of Ede1 that contains a coiled coil and a low-complexity domain. We demonstrate that Ede1 core region is essential for the endocytic function of Ede1. The core region can also promote clustering of a heterologous lipid-binding domain into discrete sites on the plasma membrane that initiate endocytic events. We propose that the clustering of the early endocytic proteins and cargo depend on phase separation mediated by Ede1.

cell biology↗

bioRxiv2019

Structural characterization of covalently stabilized human cystatin C oligomers

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10-12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from AFM, TEM and SAXS measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may provide useful to further explore the physiological relevance of different structural species of cystatin C in relationship to protein misfolding disease

biophysics↗