Hyaluronan is a natural carbohydrate polymer with a negative charge that fosters gel-like conditions crucial for its cellular functions and industrial applications. As a recognized ligand for proteins, understanding their mutual interactions provides solid ground to tune hyaluronan's gel properties using biocompatible peptides. This work employs NMR and molecular dynamics simulations to identify molecular motifs relevant to hyaluronan-peptide interactions using arginine, lysine, and glycine oligopeptides. Arginine-rich peptides exhibit the strongest binding to hyaluronan according to chemical shift perturbation measurements, followed distantly by the similarly charged lysine. This difference highlights the significance of electrostatics and the peculiarities of the guanidinium side chain in arginine, capable of non-polar interactions that further stabilize the binding. Additional nuclear Overhauser effect measurements do not show stable interaction partners, precluding strong and well-defined complexes. Finally, molecular simulations support our findings and show an extended but significant interaction region, especially for arginine, responsible for the observed enhanced binding, which can also promote cross-linking of hyaluronan polymers. Our findings pave the way for optimizing biocompatible peptides to alter hyaluronan gels' properties efficiently and also explain why hyaluronan-protein interaction typically involves positively charged arginine-rich regions also capable of forming hydrogen bonds and non-polar interactions.
- MeSH
- analýza potravin MeSH
- arginin * chemie fyziologie nedostatek MeSH
- erektilní dysfunkce etiologie farmakoterapie MeSH
- lidé MeSH
- oxid dusnatý * dějiny fyziologie nedostatek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- populární práce MeSH
Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).
- MeSH
- arginin chemie MeSH
- DNA chemická syntéza chemie MeSH
- histony chemie MeSH
- ketony chemická syntéza chemie MeSH
- nádorový supresorový protein p53 chemie MeSH
- peptidy chemie MeSH
- proteiny chemie MeSH
- reagencia zkříženě vázaná chemická syntéza chemie MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- thiminnukleotidy chemická syntéza chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36-0.62 µM for ADMA and 0.32-0.70 µM for SDMA.
We have prepared a candidate biocompatible construct for skin wound healing based on electrospun polycaprolactone (PCL) nanofibrous membranes. The membrane material was loaded either with L-arginine or with alaptide, or with a mixture of both bioactive components. Alaptide is a spirocyclic synthetic dipeptide, an analogue of melanocyte-stimulating hormone release-inhibiting factor. L-arginine is an amino acid with a basic guanidine side chain. It is a direct precursor of nitric oxide, which plays a pivotal role in skin repair. The presence and the distribution of the additives were proved with high-performance liquid chromatography, Fourier-transform infrared spectroscopy and Raman spectroscopy. The influence of L-arginine and alaptide on the morphology of the membrane was characterized using scanning electron microscopy. No statistically significant correlation between fiber diameter and drug concentration was observed. The membranes were then tested in vitro for their cytotoxicity, using primary human dermal fibroblasts, in order to obtain the optimal concentrations of the additives for in vivo tests in a rat model. The membranes with the highest concentration of L-arginine (10 wt. %) proved to be cytotoxic. The membranes with alaptide in concentrations from 0.1 to 2.5 wt.%, and with the other L-arginine concentrations (1 and 5 wt.%), did not show high toxicity. In addition, there was no observed improvement in cell proliferation on the membranes. The in vivo experiments revealed that membranes with 1.5 wt.% of alaptide or with 1.5 wt.% of alaptide in combination with 5 wt.% of L-arginine markedly accelerated the healing of skin incisions, and particularly the healing of skin burns, i.e. wounds of relatively large extent. These results indicate that our newly-developed nanofibrous membranes are promising for treating wounds with large damaged areas, where a supporting material is needed.
- MeSH
- arginin chemie MeSH
- biokompatibilní materiály chemie MeSH
- cyklické peptidy chemie MeSH
- elektrochemie MeSH
- elektrody MeSH
- fibroblasty účinky léků MeSH
- fluorescenční mikroskopie MeSH
- hojení ran účinky léků MeSH
- krysa rodu rattus MeSH
- kůže patologie MeSH
- lidé MeSH
- nanovlákna chemie MeSH
- neuropeptidy chemie MeSH
- peptidy chemie MeSH
- potkani Wistar MeSH
- proliferace buněk MeSH
- Ramanova spektroskopie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- systémy cílené aplikace léků MeSH
- techniky in vitro MeSH
- testování materiálů MeSH
- tkáňové inženýrství metody MeSH
- tkáňové podpůrné struktury chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hexameric arginine repressor, ArgR, is the feedback regulator of bacterial L-arginine regulons, and sensor of L-arg that controls transcription of genes for its synthesis and catabolism. Although ArgR function, as well as its secondary, tertiary, and quaternary structures, is essentially the same in E. coli and B. subtilis, the two proteins differ significantly in sequence, including residues implicated in the response to L-arg. Molecular dynamics simulations are used here to evaluate the behavior of intact B. subtilis ArgR with and without L-arg, and are compared with prior MD results for a domain fragment of E. coli ArgR. Relative to its crystal structure, B. subtilis ArgR in absence of L-arg undergoes a large-scale rotational shift of its trimeric subassemblies that is very similar to that observed in the E. coli protein, but the residues driving rotation have distinct secondary and tertiary structural locations, and a key residue that drives rotation in E. coli is missing in B. subtilis. The similarity of trimer rotation despite different driving residues suggests that a rotational shift between trimers is integral to ArgR function. This conclusion is supported by phylogenetic analysis of distant ArgR homologs reported here that indicates at least three major groups characterized by distinct sequence motifs but predicted to undergo a common rotational transition. The dynamic consequences of L-arg binding for transcriptional activation of intact ArgR are evaluated here for the first time in two-microsecond simulations of B. subtilis ArgR. L-arg binding to intact B. subtilis ArgR causes a significant further shift in the angle of rotation between trimers that causes the N-terminal DNA-binding domains lose their interactions with the C-terminal domains, and is likely the first step toward adopting DNA-binding-competent conformations. The results aid interpretation of crystal structures of ArgR and ArgR-DNA complexes.
- MeSH
- alosterická regulace MeSH
- arginin chemie metabolismus MeSH
- Bacillus subtilis chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- entropie MeSH
- Escherichia coli chemie genetika metabolismus MeSH
- fylogeneze MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- proteinové domény MeSH
- regulon genetika MeSH
- represorové proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
Interactions between heparin and tetraarginine in an acidic background electrolyte were investigated in capillary electrophoresis. The results showed that tetraarginine and heparin form a stable complex that migrates toward the anode immediately after coming into contact. When a zone of tetraarginine at a mg/mL concentration level passes through a zone of heparin at a μg/mL concentration level, tetraarginine is gradually removed by the formation of the complex that migrates in the opposite direction, thereby decreasing the tetraarginine peak area. The variation of the tetraarginine peak area as a function of the unfractionated heparin concentration was linear within the range 2-20 μg/mL, which enables us to detect and determine heparin concentrations undetectable with a UV detector. The same behavior was confirmed for low molecular weight heparin.
- MeSH
- arginin analýza chemie MeSH
- elektroforéza kapilární metody MeSH
- heparin analýza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polycaprolactone (PCL) was electrospun with the addition of arginine (Arg), an α-amino acid that accelerates the healing process. The efficient needleless electrospinning technique was used for the fabrication of the nanofibrous layers. The materials produced consisted mainly of fibers with diameters of between 200 and 400 nm. Moreover, both microfibers and beads were present within the layers. Higher bead sizes were observed with the increased addition of arginine. The arginine content within the layers as well as the weight of the resultant electrospun materials were enhanced with the increased addition of arginine to the electrospinning solution (1, 5 and 10 wt%). The PCL + 1% Arg nanofibrous layer contained 5.67 ± 0.04% of arginine, the PCL + 5% Arg layer 22.66 ± 0.24% of arginine and the PCL + 10% Arg layer 37.33 ± 0.39% of arginine according to the results of the elemental analysis. A high burst release within 5 h of soaking was recorded for the PCL + 5% and PCL + 10% nanofibrous layers. However, the release rate of arginine from the PCL + 1% Arg was significantly slower, reaching a maximum level after 72 h of soaking. The resulting materials were hydrophobic. Hemocompatibility testing under static conditions revealed no effect on hemolysis following the addition of arginine and the prolongation of the prothrombin time with the increased addition of arginine, thus exerting an influence on the extrinsic and common pathway of coagulation activation. The results of the dynamic hemocompatibility assessment revealed that the numbers of blood cells and platelets were not affected significantly by the various electrospun samples during incubation. The TAT, β-thromboglobulin and SC5-b9 concentrations were characterized by a moderate increase in the PCL group compared to those of the control group. The presence of arginine resulted in a decrease in the investigated hemocompatibility markers. The PMN elastase levels were comparable with respect to all the groups.
- MeSH
- arginin chemie MeSH
- biokompatibilní materiály chemie MeSH
- elektřina MeSH
- hemolýza * MeSH
- hojení ran * MeSH
- lidé MeSH
- nanovlákna chemie MeSH
- polyestery chemie MeSH
- protrombinový čas MeSH
- testování materiálů metody MeSH
- tkáňové inženýrství MeSH
- tkáňové podpůrné struktury chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination. Specialized nucleases, termed resolvases, remove (i.e., resolve) HJs. The bacterial protein RuvC is a canonical resolvase that introduces two symmetric cuts into the HJ. For complete resolution of the HJ, the two cuts need to be tightly coordinated. They are also specific for cognate DNA sequences. Using a combination of structural biology, biochemistry, and a computational approach, here we show that correct positioning of the substrate for cleavage requires conformational changes within the bound DNA. These changes involve rare high-energy states with protein-assisted base flipping that are readily accessible for the cognate DNA sequence but not for non-cognate sequences. These conformational changes and the relief of protein-induced structural tension of the DNA facilitate coordination between the two cuts. The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readouts.
- MeSH
- arginin chemie MeSH
- bakteriální proteiny chemie MeSH
- biokatalýza MeSH
- DNA bakterií chemie metabolismus MeSH
- křížová struktura DNA chemie MeSH
- párování bází MeSH
- sekvence nukleotidů MeSH
- simulace molekulární dynamiky MeSH
- Thermus thermophilus metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect.
- MeSH
- antitumorózní látky chemie MeSH
- arginin chemie MeSH
- doxorubicin analogy a deriváty chemie MeSH
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- methakryláty chemie MeSH
- myši MeSH
- nosiče léků chemie MeSH
- pilotní projekty MeSH
- polymery chemie MeSH
- rozpustnost účinky léků MeSH
- sérový albumin hovězí chemie MeSH
- uvolňování léčiv účinky léků MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
