Protein-protein interactions (PPIs) remain poorly explored targets for the treatment of Alzheimer's disease. The interaction of 14-3-3 proteins with Tau was shown to be linked to Tau pathology. This PPI is therefore seen as a potential target for Alzheimer's disease. When Tau is phosphorylated by PKA (Tau-PKA), several phosphorylation sites are generated, including two known 14-3-3 binding sites, surrounding the phosphorylated serines 214 and 324 of Tau. The crystal structures of 14-3-3 in complex with peptides surrounding these Tau phosphosites show that both these motifs are anchored in the amphipathic binding groove of 14-3-3. However, in the absence of structural data with the full-length Tau protein, the stoichiometry of the complex or the interface and affinity of the partners is still unclear. In this work, we addressed these points, using a broad range of biophysical techniques. The interaction of the long and disordered Tau-PKA protein with 14-3-3σ is restricted to two short sequences, containing phosphorylated serines, which bind in the amphipathic binding groove of 14-3-3σ. Phosphorylation of Tau is fundamental for the formation of this stable complex, and the affinity of the Tau-PKA/14-3-3σ interaction is in the 1-10 micromolar range. Each monomer of the 14-3-3σ dimer binds one of two different phosphorylated peptides of Tau-PKA, suggesting a 14-3-3/Tau-PKA stoichiometry of 2 : 1, confirmed by analytical ultracentrifugation. These results contribute to a better understanding of this PPI and provide useful insights for drug discovery projects aiming at the modulation of this interaction.
- MeSH
- Alzheimerova nemoc genetika metabolismus patologie MeSH
- exoribonukleasy chemie genetika metabolismus MeSH
- fosforylace MeSH
- konformace proteinů MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- multimerizace proteinu * MeSH
- mutace MeSH
- povrchová plasmonová rezonance MeSH
- proteinkinasy závislé na cyklickém AMP metabolismus MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- proteiny tau chemie genetika metabolismus MeSH
- serin chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The growing incidence of multidrug-resistant bacterial strains presents a major challenge in modern medicine. Antibiotic resistance is often exhibited by Staphylococcus aureus, which causes severe infections in human and animal hosts and leads to significant economic losses. Antimicrobial agents with enzymatic activity (enzybiotics) and phage therapy represent promising and effective alternatives to classic antibiotics. However, new tools are needed to study phage-bacteria interactions and bacterial lysis with high resolution and in real-time. Here, we introduce a method for studying the lysis of S. aureus at the single-cell level in real-time using atomic force microscopy (AFM) in liquid. We demonstrate the ability of the method to monitor the effect of the enzyme lysostaphin on S. aureus and the lytic action of the Podoviridae phage P68. AFM allowed the topographic and biomechanical properties of individual bacterial cells to be monitored at high resolution over the course of their lysis, under near-physiological conditions. Changes in the stiffness of S. aureus cells during lysis were studied by analyzing force-distance curves to determine Young's modulus. This allowed observing a progressive decline in cellular stiffness corresponding to the disintegration of the cell envelope. The AFM experiments were complemented by surface plasmon resonance (SPR) experiments that provided information on the kinetics of phage-bacterium binding and the subsequent lytic processes. This approach forms the foundation of an innovative framework for studying the lysis of individual bacteria that may facilitate the further development of phage therapy.
- MeSH
- bakteriofágy * MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- povrchová plasmonová rezonance MeSH
- stafylokokové infekce * MeSH
- Staphylococcus aureus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Surface plasmon resonance imaging biosensors have a number of advantages that make them superior to other analytical methods. These include the possibility of label-free detection, speed and high sensitivity to low protein concentrations. The aim of this study was to create and analyze biochips, with the help of which it is possible to test cystatin C in patient urine samples and compare the results with the one-time traditional ELISA method. The main advantage of the surface plasmon resonance imaging method is the possibility of repeated measurements over a long period of time in accordance with clinical practice. The surface of the biochip was spotted with anticystatin C and a negative control of mouse IgG at a ratio of 1:1. The aforementioned biochip was first verified using standard tests and then with patient samples, which clearly confirmed the required sensitivity even for very low concentrations of cystatin C.
- MeSH
- biosenzitivní techniky * MeSH
- cystatin C analýza moč MeSH
- lidé MeSH
- mikročipová analýza MeSH
- myši MeSH
- povrchová plasmonová rezonance * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin-proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets.
- MeSH
- analýza jednotlivých buněk metody MeSH
- kovové nanočástice chemie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- povrchová plasmonová rezonance MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- protein obsahující valosin genetika metabolismus MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- reakce na tepelný šok * MeSH
- stříbro chemie MeSH
- ubikvitin metabolismus MeSH
- vysoká teplota škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The chiral recognition of organic compounds is of vital importance in the field of pharmacology and medicine. Unfortunately, the common analytical routes used in this field are significantly restricted by time spent and equipment demands. In this work, we propose an unprecedented alternative, aimed at enantiomer discrimination and estimation of their concentrations in an uncomplicated and instantaneous manner. The proposed approach is based on the creation of an optical fiber probe with two pronounced plasmonic bands attributed to gold and silver. The gold or silver surfaces were grafted with moieties, able to enunciating entrap chiral amines from solution, resulting in a wavelength shift corresponding to each plasmonic metal. As a model compound of chiral amine, we chose the DOPA, also taking in mind its high medical relevancy. For chiral detection, the optical fiber probe was simply immersed in an analytical solution of DOPA, and the selective shift of gold or silver plasmon bands was observed in the reflected light depending on DOPA chirality. The observed shifts depend on the concentration of DOPA enantiomers. In the case of a racemic mixture, the shifts of both plasmonic bands emerge, making possible the simultaneous determination of enantiomer concentrations and their ratio. The analytical cycle takes several minutes and requires very simple laboratory equipment.
In early stages of Alzheimer's disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ1-40, Aβ1-42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ1-40 and Aβ1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ1-40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aβ1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.
- MeSH
- 17-hydroxysteroidní dehydrogenasy chemie genetika MeSH
- Alzheimerova nemoc diagnóza genetika patologie MeSH
- amyloidní beta-protein chemie MeSH
- biosenzitivní techniky metody MeSH
- ionty chemie MeSH
- lidé MeSH
- mitochondriální proteiny chemie MeSH
- mitochondrie chemie MeSH
- peptidové fragmenty chemie genetika MeSH
- peptidylprolylisomerasa F chemie genetika MeSH
- povrchová plasmonová rezonance metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Progressive mitochondrial dysfunction due to the accumulation of amyloid beta (Aβ) peptide within the mitochondrial matrix represents one of the key characteristics of Alzheimer's disease (AD) and appears already in its early stages. Inside the mitochondria, Aβ interacts with a number of biomolecules, including cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10), and affects their physiological functions. However, despite intensive ongoing research, the exact mechanisms through which Aβ impairs mitochondrial functions remain to be explained. In this work, we studied the interactions of Aβ with cypD and 17β-HSD10 in vitro using the surface plasmon resonance (SPR) method and determined the kinetic parameters (association and dissociation rates) of these interactions. This is the first work which determines all these parameters under the same conditions, thus, enabling direct comparison of relative affinities of Aβ to its mitochondrial binding partners. Moreover, we used the determined characteristics of the individual interactions to simulate the concurrent interactions of Aβ with cypD and 17β-HSD10 in different model situations associated with the progression of AD. This study not only advances the understanding of Aβ-induced processes in mitochondria during AD, but it also provides a new perspective on research into complex multi-interaction biomolecular processes in general.
- MeSH
- 17-hydroxysteroidní dehydrogenasy chemie metabolismus MeSH
- Alzheimerova nemoc metabolismus MeSH
- amyloidní beta-protein chemie metabolismus MeSH
- biosenzitivní techniky MeSH
- lidé MeSH
- mitochondriální proteiny chemie metabolismus MeSH
- peptidylprolylisomerasa F chemie metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Our recent experience of the COVID-19 pandemic has highlighted the importance of easy-to-use, quick, cheap, sensitive and selective detection of virus pathogens for the efficient monitoring and treatment of virus diseases. Early detection of viruses provides essential information about possible efficient and targeted treatments, prolongs the therapeutic window and hence reduces morbidity. Graphene is a lightweight, chemically stable and conductive material that can be successfully utilized for the detection of various virus strains. The sensitivity and selectivity of graphene can be enhanced by its functionalization or combination with other materials. Introducing suitable functional groups and/or counterparts in the hybrid structure enables tuning of the optical and electrical properties, which is particularly attractive for rapid and easy-to-use virus detection. In this review, we cover all the different types of graphene-based sensors available for virus detection, including, e.g., photoluminescence and colorimetric sensors, and surface plasmon resonance biosensors. Various strategies of electrochemical detection of viruses based on, e.g., DNA hybridization or antigen-antibody interactions, are also discussed. We summarize the current state-of-the-art applications of graphene-based systems for sensing a variety of viruses, e.g., SARS-CoV-2, influenza, dengue fever, hepatitis C virus, HIV, rotavirus and Zika virus. General principles, mechanisms of action, advantages and drawbacks are presented to provide useful information for the further development and construction of advanced virus biosensors. We highlight that the unique and tunable physicochemical properties of graphene-based nanomaterials make them ideal candidates for engineering and miniaturization of biosensors.
- MeSH
- Betacoronavirus genetika izolace a purifikace patogenita MeSH
- biosenzitivní techniky * přístrojové vybavení metody trendy MeSH
- design vybavení MeSH
- DNA virů analýza genetika MeSH
- elektrochemické techniky MeSH
- grafit * chemie MeSH
- hybridizace nukleových kyselin MeSH
- klinické laboratorní techniky * přístrojové vybavení metody statistika a číselné údaje MeSH
- kolorimetrie MeSH
- koronavirové infekce diagnóza epidemiologie virologie MeSH
- kvantové tečky chemie MeSH
- lidé MeSH
- luminiscence MeSH
- nanostruktury chemie MeSH
- pandemie MeSH
- povrchová plasmonová rezonance MeSH
- Ramanova spektroskopie MeSH
- reakce antigenu s protilátkou MeSH
- virologie metody MeSH
- virová pneumonie diagnóza epidemiologie virologie MeSH
- viry genetika izolace a purifikace patogenita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s-1, kd 5.8 × 104 s-1, and KD 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10-cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.
- MeSH
- 17-hydroxysteroidní dehydrogenasy mozkomíšní mok metabolismus MeSH
- Alzheimerova nemoc metabolismus MeSH
- amyloidový prekurzorový protein beta genetika MeSH
- kinetika MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- mozek metabolismus MeSH
- peptidylprolylisomerasa F metabolismus MeSH
- potkani transgenní MeSH
- potkani Wistar MeSH
- povrchová plasmonová rezonance MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed β-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- fruktosa metabolismus MeSH
- kalorimetrie MeSH
- krystalografie rentgenová MeSH
- lektiny chemie genetika metabolismus MeSH
- Photorhabdus metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
