Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.
- MeSH
- Leukemia, Myeloid, Acute * genetics metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoplasm metabolism MeSH
- Nuclear Localization Signals metabolism MeSH
- Nuclear Proteins * genetics metabolism MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Mutation * MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 * metabolism genetics chemistry MeSH
- Nucleophosmin MeSH
- Nuclear Export Signals MeSH
- Protein Transport MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The aim of the study is to investigate the differences in the interaction of three structurally diverse anthocyanidins, namely peonidin, petunidin, and delphinidin, as well as their glucosides with model biological membranes, human albumin, and plasmid DNA in order to look into their structure-activity relationships. Fluorimetric studies, as well as ATR-FTIR analyses, were jointly used in order to determine the changes observed in both the hydrophilic and hydrophobic layers of cell-mimic membranes (MM) which reflected the membrane lipid composition of tumour cells and red blood cell membranes (RBCM). Our results showed that anthocyanins and anthocyanidins can cause an increase in the packing order of the polar heads of lipids, as well as interact with their deeper layers by reducing the fluidity of lipid chains. The results presented here indicate that all compounds tested here possessed the ability to bind to human serum albumin (HSA) and the presence of a glucose molecule within the structures formed by anthocyanidin reduces their ability to bind to proteins. Using fluorescence correlation spectroscopy, it was demonstrated that the compounds tested here were capable of forming stable complexes with plasmid DNA and, particularly, strong DNA conformational changes were observed in the presence of petunidin and corresponding glucoside, as well as delphinidin. The results we obtained can be useful in comprehending the anthocyanins therapeutic action as molecular antioxidants and provide a valuable insight into their mechanism of action.
Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.
- MeSH
- Chlorophyll chemistry genetics radiation effects MeSH
- Fluorescence MeSH
- Spectrometry, Fluorescence MeSH
- Photosynthesis genetics MeSH
- Photosystem II Protein Complex genetics radiation effects MeSH
- Phototrophic Processes genetics MeSH
- Antennapedia Homeodomain Protein chemistry genetics MeSH
- Hydrogen-Ion Concentration MeSH
- Protein Aggregates genetics MeSH
- Cluster Analysis MeSH
- Light adverse effects MeSH
- Light-Harvesting Protein Complexes chemistry genetics MeSH
- Thylakoids chemistry genetics radiation effects MeSH
- Zeaxanthins genetics MeSH
- Publication type
- Journal Article MeSH
Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.
- MeSH
- Ion Transport MeSH
- Protein Conformation MeSH
- Fatty Acids metabolism MeSH
- Membrane Potential, Mitochondrial MeSH
- Mitochondria metabolism MeSH
- Mice MeSH
- Protons * MeSH
- Adenine Nucleotide Translocator 1 chemistry metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.
- MeSH
- Bardet-Biedl Syndrome genetics metabolism pathology MeSH
- Cell Line MeSH
- Cilia metabolism MeSH
- CRISPR-Cas Systems genetics MeSH
- Cytoplasm metabolism MeSH
- Gene Editing MeSH
- Microscopy, Fluorescence MeSH
- Fluorescence Recovery After Photobleaching MeSH
- Humans MeSH
- Mutation MeSH
- Protein Subunits genetics metabolism MeSH
- Microtubule-Associated Proteins deficiency genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.
- MeSH
- Anti-Bacterial Agents chemistry isolation & purification pharmacology MeSH
- Cell Membrane chemistry drug effects MeSH
- Ethanolamines chemistry MeSH
- Drug Combinations MeSH
- Fluorescent Dyes chemistry MeSH
- Spectrometry, Fluorescence MeSH
- Phosphatidylcholines chemistry MeSH
- Phosphatidylethanolamines chemistry MeSH
- Phosphatidylglycerols chemistry MeSH
- Antimicrobial Cationic Peptides chemistry isolation & purification pharmacology MeSH
- Skin chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Magainins chemistry isolation & purification pharmacology MeSH
- Xenopus Proteins chemistry isolation & purification pharmacology MeSH
- Boron Compounds chemistry MeSH
- Drug Synergism MeSH
- Protein Binding MeSH
- Xenopus laevis MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Spheroids-three-dimensional aggregates of cells grown from a cancer cell line-represent a model of living tissue for chemotherapy investigation. Distribution of chemotherapeutics in spheroid sections was determined using the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Proliferating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal fluorescence microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM only data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images. To allow comparison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes or diameters, we introduced a common diffusion-related coordinate, the distance from the spheroid boundary. In a procedure referred to as "peeling", we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. This novel approach makes it possible to differentiate between peripheral cells that died due to therapy and the innermost cells which died naturally. Two novel algorithms-for MALDI MS image denoising and for weighting of MALDI MSI and LSCM data by the presence of cell nuclei-are also presented.
- MeSH
- Spheroids, Cellular drug effects MeSH
- Microscopy, Confocal methods MeSH
- Humans MeSH
- Neoplasms drug therapy MeSH
- Antineoplastic Agents pharmacokinetics pharmacology MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Models, Theoretical MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as β-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.
- MeSH
- Anti-Bacterial Agents chemistry metabolism MeSH
- Phosphatidylcholines chemistry MeSH
- Phosphatidylglycerols chemistry MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Antimicrobial Cationic Peptides chemistry metabolism MeSH
- Kinetics MeSH
- Protein Conformation, alpha-Helical MeSH
- Protein Conformation, beta-Strand MeSH
- Lipid Bilayers chemistry MeSH
- Permeability MeSH
- Amino Acid Sequence MeSH
- Molecular Dynamics Simulation MeSH
- Publication type
- Journal Article MeSH
DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.
- MeSH
- Tumor Suppressor p53-Binding Protein 1 metabolism MeSH
- Microscopy, Confocal methods MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Recombinational DNA Repair * MeSH
- Protein Transport MeSH
- Single Molecule Imaging methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.
- MeSH
- Alzheimer Disease enzymology MeSH
- Amino Acids antagonists & inhibitors MeSH
- HEK293 Cells MeSH
- Heterocyclic Compounds, 2-Ring pharmacology MeSH
- Kinetics MeSH
- Picolinic Acids pharmacology MeSH
- Humans MeSH
- Drug Evaluation, Preclinical methods MeSH
- Pyrimidinones pharmacology MeSH
- Amyloid Precursor Protein Secretases antagonists & inhibitors metabolism MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
