The coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years. Because of the relatively well-understood principles that determine the properties of coiled coil peptides and proteins, macromolecular systems containing the coiled coil motif have been suggested for various applications. This short review focuses on hybrid polymer coiled coil systems designed for drug delivery purposes. After a short introduction, the most important features of the coiled coils (stability, association number, oligomerization selectivity and orientation of helices) are described, and the factors influencing these characteristics are discussed. Several examples of the most interesting biomedical applications of the polymer-coiled coil systems (according to the authors' opinion) are presented.

• MeSH
• aminokyselinové motivy * MeSH
• hydrogely MeSH
• konformace proteinů MeSH
• lékové transportní systémy metody   MeSH
• molekulární modely MeSH
• rekombinantní proteiny aplikace a dávkování   genetika   farmakologie   MeSH
• rozpustnost MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

A two-component CD20 (non-internalizing) receptor crosslinking system based on the biorecognition of complementary coiled-coil forming peptides was evaluated. Exposure of B cells to Fab'-peptide1 conjugate decorates the cell surface with peptide1; further exposure of the decorated cells to P-(peptide2)x (P is the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone) results in the formation of coiled-coil heterodimers at the cell surface with concomitant induction of apoptosis. The aim of this study was to determine the potential immunogenicity of this therapeutic system that does not contain low molecular weight drugs. Enantiomeric peptides (L- and D-CCE and L- and D-CCK), HPMA copolymer-peptide conjugates, and Fab' fragment-peptide conjugates were synthesized and the immunological properties of peptide conjugates evaluated in vitro on RAW264.7 macrophages and in vivo on immunocompetent BALB/c mice. HPMA copolymer did not induce immune response in vitro and in vivo. Administration of P-peptide conjugates with strong adjuvant resulted in antibody response directed to the peptide. Fab' was responsible for macrophage activation of Fab'-peptide conjugates and a major factor in the antibody induction following i.v. administration of Fab'-conjugates. There was no substantial difference in the ability of conjugates of D-peptides and conjugates of L-peptides to induce Ab response.

• MeSH
• akrylamidy aplikace a dávkování   chemie   imunologie   MeSH
• buněčné linie MeSH
• imunoglobuliny - Fab fragmenty aplikace a dávkování   chemie   imunologie   MeSH
• makrofágy účinky léků   imunologie   MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidy aplikace a dávkování   chemie   imunologie   MeSH
• sekvence aminokyselin MeSH
• T-lymfocyty účinky léků   imunologie   MeSH
• tvorba protilátek účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.

• MeSH
• methakryláty chemie   MeSH
• nosiče léků chemie   MeSH
• oligopeptidy chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• techniky syntézy na pevné fázi MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.

• MeSH
• akrylamidy chemická syntéza   farmakologie   MeSH
• biokompatibilní materiály chemická syntéza   farmakologie   MeSH
• cytostatické látky chemie   farmakologie   MeSH
• doxorubicin chemie   farmakologie   MeSH
• leukemie farmakoterapie   MeSH
• methakryláty chemie   MeSH
• monoklonální protilátky chemie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nosiče léků chemie   MeSH
• polymery chemie   farmakologie   MeSH
• proliferace buněk MeSH
• protinádorová antibiotika chemie   farmakologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.

• MeSH
• akrylamidy chemie   MeSH
• antigeny nádorové imunologie   metabolismus   MeSH
• bakteriální transformace MeSH
• cirkulární dichroismus MeSH
• click chemie metody   MeSH
• dimerizace MeSH
• ELISA MeSH
• Escherichia coli MeSH
• imunokonjugáty chemie   imunologie   farmakologie   MeSH
• karboanhydrasy imunologie   metabolismus   MeSH
• karcinom farmakoterapie   enzymologie   imunologie   patologie   MeSH
• klonování DNA MeSH
• lidé MeSH
• molekulární konformace MeSH
• monoklonální protilátky chemie   genetika   imunologie   MeSH
• nádorové biomarkery imunologie   metabolismus   MeSH
• nosiče léků chemická syntéza   farmakologie   MeSH
• oligopeptidy chemická syntéza   imunologie   farmakologie   MeSH
• plazmidy MeSH
• polyethylenglykoly chemie   MeSH
• rekombinantní proteiny chemie   genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

• MeSH
• agregace trombocytů MeSH
• batroxobin metabolismus   MeSH
• bodová mutace MeSH
• časové faktory MeSH
• dospělí MeSH
• fibrin metabolismus   MeSH
• fibrinogen genetika   metabolismus   MeSH
• fibrinolýza MeSH
• hemokoagulace MeSH
• heterozygot MeSH
• konformace proteinů MeSH
• lidé MeSH
• samovolný potrat krev   MeSH
• těhotenství MeSH
• trombin metabolismus   MeSH
• trombofilie krev   genetika   MeSH
• vyšetření krevní srážlivosti MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bβ chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bβ chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.

• Publikační typ
• časopisecké články MeSH

Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into typical 10-12 nm wide filaments. The primary structure of these proteins includes the signature central 'rod' domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A11 tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A11 tetramer, flexibility of linkers L1 and L12 enables 'backfolding' of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.

• MeSH
• cytoskelet * metabolismus   MeSH
• intermediární filamenta * metabolismus   MeSH
• molekulární struktura MeSH
• sekvence aminokyselin MeSH
• vimentin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.

• MeSH
• fibrinogen chemie   metabolismus   MeSH
• lidé MeSH
• posttranslační úpravy proteinů * MeSH
• sekundární struktura proteinů MeSH
• simulace molekulární dynamiky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between structure and anti-ROCK2 activity in a group of purine derivatives substituted at the C6 atom by piperidin-1-yl or azepan-1-yl groups. Structure-activity relationship (SAR) analyses suggested that anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds at the C2 atom or by expansion of the C6 side chain. These inhibitors of ROCK can reach effective concentrations within cells, as demonstrated by a decrease in phosphorylation of the ROCK target MLC, and by inhibition of the ROCK-dependent invasion of melanoma cells in the collagen matrix. Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of other sensitive kinases identified by the screening of a broad panel of protein kinases.

• MeSH
• inhibitory proteinkinas chemická syntéza   farmakologie   MeSH
• kinázy asociované s rho antagonisté a inhibitory   MeSH
• lidé MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• pohyb buněk účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky chemická syntéza   farmakologie   MeSH
• puriny chemická syntéza   farmakologie   MeSH
• signální transdukce účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH