The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.

• MeSH
• Actin Capping Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Phosphatidylinositol Phosphates chemistry   metabolism   MeSH
• Phylogeny MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Chickens MeSH
• Phosphatidic Acids chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Mutation MeSH
• Arabidopsis Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Avian Proteins antagonists & inhibitors   chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Protein Binding MeSH
• Computational Biology MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

• MeSH
• Molecular Biology MeSH
• Proteins MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• biochemie
• molekulární biologie, molekulární medicína

Diagnostikování astmatu je někdy obtížné, zvláště u malých dětí nespolupracujících při spirometrii. Hladina eozinofilního kationického proteinu (ECP) pomáhá při diagnóze astmatu, jehož podkladem je tzv. eozinofilní typ zánětu dýchacích cest. ECP je produkován aktivovanými eozinofily, jejichž množství a vlastnosti jsou u atopiků změněné. Hladina ECP v séru koreluje s intenzitou zánětu a je tedy možné ji využít jako ukazatel závažnosti astmatu. Existují však určité problémy technické a interpretační při použití tohoto ukazatele.Našim cílem bylo zjistit, zda ECP je dobrým ukazatelem pro diagnostiku a léčbu astmatu, zda koreluje s klinickými potížemi a spirometrií a jaké jsou rozdíly hladin u atopického a neatopického astmatu.Soubor 43 astmatiků byl vyšetřen alergologicky, spirometricky a opakovaně vyšetřen ECP.Hladina ECP byla vyšetřena CAP systémem Pharmacia. Zvýšení ECP bylo častejší u atopického (71%) než u neatopického (20%) astmatu. Hladina ECP korelovala s počtem eozinofilů v perif. krvi a celk. IgE, ale ne s potížemi a spirometrií. Mezi astmatiky s atopií byla skupina dětí, jejichž astma bylo komplikované, které ale měly normální ECP. ECP je dobrým ukazatelem léčby astmatu, ale jako diagnostický ukazatel je nutno jej interpretovat individuálně.

The diagnosis of bronchial asthma is sometimes difficult, especially in little children, who are not able to perform spirometry correctly. Examination of eosinophil cationic protein (ECP) can help in diagnosis of astma, which is known to be the eosinophilic inflammation of airways. ECP is produced by activated eosinophils. Their amount and properties are changed in atopic individuals. It is known, that the level of ECP in serum correlates with the intensity of the inflammation and therefore lends itself for use as a marker of asthma severity. There are, however, certain technical and interpretative difficulties in its use as such marker.The aim was to find if ECP is a good marker for therapy, if there is correlation between ECP level, clinical symptoms and spirometry and what is the difference in atopic versus non atopic asthma.In a group of 43 asthmatic children, allergologic, spirometric and repeated ECP examination were performed. The ECP level was determined by Pharmacia CAP system. Elevated level of ECP was more often found in atopic (71%) then in nonatopic ( 20%) children. Correlation was found between ECP and blood eosinophil count and total IgE level, but not with symptoms and spirometry. There is a small group of children among atopic patients with relatively complicated asthma, whose ECP level is normal. ECP is a good marker for therapy. As diagnostic marker, it must be interpreted individually.

• MeSH
• Asthma diagnosis   etiology   drug therapy   MeSH
• Hypersensitivity, Immediate diagnosis   MeSH
• Child MeSH
• Eosinophils diagnostic use   blood   secretion   MeSH
• Immunoglobulin E blood   MeSH
• Humans MeSH
• Spirometry MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Review MeSH
• Comparative Study MeSH

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

• MeSH
• COVID-19 virology   MeSH
• Humans MeSH
• Methyltransferases analysis   genetics   metabolism   MeSH
• Antibodies, Monoclonal analysis   MeSH
• RNA Caps genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• SARS-CoV-2 chemistry   enzymology   genetics   MeSH
• Protein Transport MeSH
• Viral Nonstructural Proteins analysis   genetics   metabolism   MeSH
• Viral Regulatory and Accessory Proteins analysis   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The effect of non-denaturing concentrations of three different organic solvents, formamide, acetone and isopropanol, on the structure of haloalkane dehalogenases DhaA, LinB, and DbjA at the protein-solvent interface was studied using molecular dynamics simulations. Analysis of B-factors revealed that the presence of a given organic solvent mainly affects the dynamical behavior of the specificity-determining cap domain, with the exception of DbjA in acetone. Orientation of organic solvent molecules on the protein surface during the simulations was clearly dependent on their interaction with hydrophobic or hydrophilic surface patches, and the simulations suggest that the behavior of studied organic solvents in the vicinity of hyrophobic patches on the surface is similar to the air/water interface. DbjA was the only dimeric enzyme among studied haloalkane dehalogenases and provided an opportunity to explore effects of organic solvents on the quaternary structure. Penetration and trapping of organic solvents in the network of interactions between both monomers depends on the physico-chemical properties of the organic solvents. Consequently, both monomers of this enzyme oscillate differently in different organic solvents. With the exception of LinB in acetone, the structures of studied enzymes were stabilized in water-miscible organic solvents.

• MeSH
• 2-Propanol chemistry   pharmacology   MeSH
• Acetone chemistry   pharmacology   MeSH
• Formamides chemistry   pharmacology   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Hydrolases chemistry   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary drug effects   MeSH
• Models, Molecular MeSH
• Solvents chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Structure, Tertiary drug effects   MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mpox is a zoonotic disease caused by the mpox virus (MPXV), which has gained attention due to its rapid and widespread transmission, with reports from more than 100 countries. The virus belongs to the Orthopoxvirus genus, which also includes variola virus and vaccinia virus. In poxviruses, the RNA cap is crucial for the translation and stability of viral mRNAs and also for immune evasion. This study presents the crystal structure of the mpox 2'-O-methyltransfarase VP39 in complex with a short cap-0 RNA. The RNA substrate binds to the protein without causing any significant changes to its overall fold and is held in place by a combination of electrostatic interactions, π-π stacking and hydrogen bonding. The structure also explains the mpox VP39 preference for a guanine base at the first position; it reveals that guanine forms a hydrogen bond that an adenine would not be able to form.

• MeSH
• Humans MeSH
• Methyltransferases chemistry   MeSH
• Methylation MeSH
• Mpox, Monkeypox * MeSH
• RNA Caps * metabolism   MeSH
• Binding Sites MeSH
• Viral Proteins genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.

• MeSH
• Microscopy, Atomic Force MeSH
• Surface Properties MeSH
• Proteins chemistry   MeSH
• Spectrum Analysis methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• DNA Damage MeSH
• Telomeric Repeat Binding Protein 2 * MeSH
• Telomere-Binding Proteins * metabolism   MeSH
• Proteomics MeSH
• Shelterin Complex * MeSH
• Telomere metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVE: The aberrant expression of myeloid antigens on acute lymphoblastic leukemia (ALL) cells is a well-documented phenomenon. So far, there have been no reports of a functional consequence of this aberrant expression. The granulocytic marker carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6, CD66c) is a GPI-anchored molecule that is reported to be the most frequently aberrantly expressed myeloid marker in ALL with a strong correlation with genotype. MATERIALS AND METHODS: We mimicked CEACAM6 signaling in ALL cells by cross-linking with anti-CEACAM6 antibody. Next, we measured a response to CEACAM6 signaling by integrin subunits expression, integrin ligand binding, phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), Akt, and p38 mitogen-activated protein kinase (MAPK) and apoptosis by flow cytometry. RESULTS: Following CEACAM6 cross-linking in ALL cells, we detected Erk1/2, Akt, and p38 MAPK phosphorylation and integrin upregulation, as well as enhanced binding of integrin ligands (vascular cell adhesion molecule-1 [VCAM-1] and intercellular cell adhesion molecule-1 [ICAM-1]). However, CEACAM6 signaling resulted in an increase in apoptosis, unlike other GPI-anchored molecules, such as CD24. CONCLUSION: The present study is the first to demonstrate the functional consequences of CEACAM6 cross-linking in B-cell precursor ALL cells.

• MeSH
• Antigens, Neoplasm immunology   metabolism   MeSH
• Apoptosis MeSH
• Antigens, CD immunology   metabolism   MeSH
• Phosphorylation drug effects   immunology   MeSH
• GPI-Linked Proteins MeSH
• Immunologic Capping MeSH
• Integrins immunology   metabolism   MeSH
• Humans MeSH
• MAP Kinase Signaling System MeSH
• Intercellular Adhesion Molecule-1 immunology   metabolism   MeSH
• Mitogen-Activated Protein Kinase 3 immunology   metabolism   MeSH
• p38 Mitogen-Activated Protein Kinases immunology   metabolism   MeSH
• Cell Adhesion Molecules antagonists & inhibitors   immunology   metabolism   MeSH
• Cell Line, Tumor MeSH
• Oncogene Protein v-akt immunology   metabolism   MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma immunology   metabolism   MeSH
• Antibodies, Neoplasm immunology   pharmacology   MeSH
• Gene Expression Regulation, Leukemic MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5' end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.

• MeSH
• Circovirus genetics   immunology   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Immunization MeSH
• Molecular Sequence Data MeSH
• Swine Diseases diagnosis   prevention & control   virology   MeSH
• Swine virology   MeSH
• Antibodies, Viral blood   MeSH
• Recombinant Proteins genetics   immunology   metabolism   MeSH
• Amino Acid Sequence MeSH
• Porcine Postweaning Multisystemic Wasting Syndrome diagnosis   prevention & control   virology   MeSH
• Virion metabolism   MeSH
• Capsid Proteins diagnostic use   genetics   immunology   metabolism   MeSH
• Viral Vaccines administration & dosage   immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH