Covalent modification Dotaz Zobrazit nápovědu
- Publikační typ
- abstrakt z konference MeSH
The covalent reversible modification of proteins is a validated strategy for the development of probes and candidate therapeutics. However, the covalent reversible targeting of noncatalytic lysines is particularly challenging. Herein, we characterize the 2-hydroxy-1-naphthaldehyde (HNA) fragment as a targeted covalent reversible ligand of a noncatalytic lysine (Lys720) of the Krev interaction trapped 1 (KRIT1) protein. We show that the interaction of HNA with KRIT1 is highly specific, results in prolonged residence time of >8 h, and inhibits the Heart of glass 1 (HEG1)-KRIT1 protein-protein interaction (PPI). Screening of HNA derivatives identified analogs exhibiting similar binding modes as the parent fragment but faster target engagement and stronger inhibition activity. These results demonstrate that HNA is an efficient site-directing fragment with promise in developing HEG1-KRIT1 PPI inhibitors. Further, the aldimine chemistry, when coupled with templating effects that promote proximity, can produce a long-lasting reversible covalent modification of noncatalytic lysines.
- Publikační typ
- časopisecké články MeSH
This mini-review summarizes the development of the derivatives and conjugates of Amphotericin B (AMB) which have not been clinically applied so far but are therapeutically promising. Effects of chemical modifications of AMB upon biological activities of the resulting derivatives are discussed. The examples presented include variation of functional groups in AMB, covalent conjugates with other molecules, poly(ethylene glycols) and polysaccharides. Possibilities of targeted delivery of AMB are discussed.
Polydopamine (PDA), also known as synthetic melanin, is widely used as a biomimetic anchoring layer for the modification of various solid substrates. PDA is utilized for a wide range of biomedical, sensing and tribological applications, even though the polymer's precise covalent structure has not been completely revealed yet. Even more, it is not evident to which extent the chemical nature of the substrate, on which the layer is formed, influences and predetermines the covalent structure of resulting PDA. In this contribution, we have studied the growth of PDA using various surface-sensitive techniques such as spectroscopic ellipsometry, atomic force microscopy and X-ray photoelectron spectroscopy. We supplemented grazing angle attenuated total reflection FTIR spectroscopy with multivariate statistical analysis to further gain analytical power. We have particularly focused on the effects of polymerization time and substrate on the PDA structure. We found notable differences in the chemical composition of PDA formed on gold and on surfaces terminated with oxides/reactive hydroxides such as silicon and N-dopped-TiO2 in the early stages of the layer formation. At the later stages of layer formation, a merely unified chemical structure was observed independently on the type of substrate.
- MeSH
- indoly * MeSH
- polymerizace MeSH
- polymery * MeSH
- povrchové vlastnosti MeSH
- Publikační typ
- časopisecké články MeSH
We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9 m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.
Cytochrome c (cyt c), in addition to its function as an electron shuttle in respiratory chain, is able to perform as a pseudo-peroxidase with a critical role during apoptosis. Incubation of cyt c with an excess of hydrogen peroxide leads to a suicide inactivation of the protein, which is accompanied by heme destruction and covalent modification of numerous amino acid residues. Although steady-state reactions of cyt c with an excess of hydrogen peroxide represent non-physiological conditions, they might be used for analysis of the first-modified amino acid in in vivo. Here, we observed oxidation of tyrosine residues 67 and 74 and heme as the first modifications found upon incubation with hydrogen peroxide. The positions of the oxidized tyrosines suggest a possible migration pathway of hydrogen peroxide-induced radicals from the site of heme localization to the protein surface. Analysis of a size of folded fraction of cyt c upon limited incubation with hydrogen peroxide indicates that the early oxidation of amino acids triggers an accelerated destruction of cyt c. Position of channels from molecular dynamics simulation structures of cyt c points to a location of amino acid residues exposed to reactive oxidants that are thus more prone to covalent modification.
- MeSH
- cirkulární dichroismus MeSH
- cytochromy c chemie genetika metabolismus MeSH
- hmotnostní spektrometrie MeSH
- koně MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- oxidace-redukce MeSH
- peroxid vodíku farmakologie MeSH
- proteolýza MeSH
- simulace molekulární dynamiky MeSH
- stabilita proteinů MeSH
- tyrosin chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396-His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp-His adduct mainly for binding and to a smaller extent for electron transfer.
- MeSH
- bilirubin metabolismus MeSH
- Hypocreales metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely * MeSH
- oxidace-redukce MeSH
- oxidoreduktasy působící na CH-CH vazby metabolismus MeSH
- transport elektronů fyziologie MeSH
- vazba proteinů fyziologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study was aimed at verifying the hypothesis that acute kidney failure accompanying cisplatin administration in the cancer therapy could be due to cisplatin interaction with the cytoplasmic part of Na(+)/K(+)-ATPase. Our results demonstrated that cisplatin-binding caused inhibition of Na(+)/K(+)-ATPase, in contrast to other platinated chemotherapeutics such as carboplatin and oxaliplatin, which are known to be much less nephrotoxic. To acquire more detailed structural information, we performed a series of experiments with the isolated large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45 loop) of Na(+)/K(+)-ATPase. Electrochemistry showed that cisplatin is bound to the cysteine residues of the C45 loop, mass spectrometry revealed a modification of the C45 peptide fragment GSHMASLEAVETLGSTSTICSDK, which contains the conserved phosphorylated residue Asp369. Hence, we hypothesize that binding of cisplatin to Cys367 can cause sterical obstruction during the phosphorylation or dephosphorylation step of the Na(+)/K(+)-ATPase catalytic cycle.
- MeSH
- cisplatina metabolismus farmakologie MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- mozková kůra enzymologie MeSH
- prasata MeSH
- protinádorové látky metabolismus farmakologie MeSH
- sodíko-draslíková ATPasa antagonisté a inhibitory metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.
- MeSH
- buněčné linie MeSH
- cirkulární dichroismus MeSH
- cystein genetika MeSH
- dánio pruhované MeSH
- down regulace MeSH
- isoindoly farmakologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- mozek metabolismus MeSH
- mutageneze cílená MeSH
- organoselenové sloučeniny farmakologie MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- proteiny dánia pruhovaného chemie metabolismus MeSH
- stabilita proteinů účinky léků MeSH
- tyrosin-3-monooxygenasa metabolismus MeSH
- vazba proteinů účinky léků MeSH
- vazebná místa účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
