Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75 000 and 525 000 M-1 s-1 for HDAC7 and HDAC5, respectively. Results reported here could help in developing class IIa HDACs inhibitors and also in the search for new natural class IIa HDACs substrates.
To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.
- MeSH
- Adamantane analogs & derivatives chemistry MeSH
- Aryl Hydrocarbon Hydroxylases chemistry ultrastructure MeSH
- Models, Chemical MeSH
- Financing, Organized MeSH
- Microscopy, Fluorescence methods MeSH
- Photochemistry methods MeSH
- Protein Conformation MeSH
- Models, Molecular MeSH
- Computer Simulation MeSH
- Substrate Specificity MeSH
- Protein Binding MeSH
- Binding Sites MeSH
BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.
- MeSH
- beta-N-Acetylhexosaminidases chemistry metabolism MeSH
- Phylogeny MeSH
- Glycosylation MeSH
- Catalytic Domain MeSH
- Kinetics MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Reproducibility of Results MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid MeSH
- Molecular Dynamics Simulation MeSH
- Substrate Specificity MeSH
- Talaromyces enzymology MeSH
- Computational Biology * MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Biodegradable nanoparticles based on stearic acid-modified poly(glycerol adipate) (PGAS) are promising carriers for drug delivery. In order to investigate the impact of the particle interface characteristics on the biological fate, PGAS nanoparticles are covalently and noncovalently coated with N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. HPMA copolymer-modified PGAS nanoparticles have similar particle sizes, but less negative zeta-potentials. Nanoparticles are double labeled with the fluorescent dyes DiR (noncovalently) and DYOMICS-676 (covalently bound to HPMA copolymer), and their biodistribution is investigated noninvasively by multispectral optical imaging. Both covalent and noncovalent coatings cause changes in the pharmacokinetics and biodistribution in healthy and tumor-bearing mice. In addition to the intended tumor accumulation, high signals of both fluorescent dyes are also observed in other organs, including liver, ovaries, adrenal glands, and bone. The unintended accumulation of nanocarriers needs further detailed and systematic investigations, especially with respect to the observed ovarian and adrenal gland accumulation.
- MeSH
- Biodegradable Plastics chemistry MeSH
- HT29 Cells MeSH
- Fluorescent Dyes chemistry MeSH
- Drug Delivery Systems * MeSH
- Humans MeSH
- Methacrylates administration & dosage chemistry MeSH
- Mice MeSH
- Neoplasms drug therapy genetics pathology MeSH
- Nanoparticles administration & dosage chemistry MeSH
- Drug Carriers administration & dosage chemistry MeSH
- Polyesters administration & dosage chemistry MeSH
- Tissue Distribution drug effects MeSH
- Xenograft Model Antitumor Assays MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP (C5'-O5'-C4'-O4'), cocCTP (C5'-O5'-C4'-C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP-substrate, 2dCTP-poor substrate, coCTP-chain terminator, cocCTP-inhibitor). Moreover, extremely rare events-as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement-were captured.
- MeSH
- Cytidine Triphosphate analogs & derivatives metabolism MeSH
- Caliciviridae Infections virology MeSH
- Humans MeSH
- Norovirus enzymology metabolism MeSH
- RNA-Dependent RNA Polymerase metabolism MeSH
- Molecular Dynamics Simulation MeSH
- Molecular Docking Simulation MeSH
- Substrate Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.
Squaramate-linked 2'-deoxycytidine 5'-O-triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate-linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys-containing peptides. Squaramate-linked DNA formed covalent cross-links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.
- MeSH
- DNA metabolism MeSH
- Humans MeSH
- Lysine metabolism MeSH
- Nucleotides metabolism MeSH
- Peptides chemistry MeSH
- Proteins chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Arginine chemistry MeSH
- DNA chemical synthesis chemistry MeSH
- Histones chemistry MeSH
- Ketones chemical synthesis chemistry MeSH
- Tumor Suppressor Protein p53 chemistry MeSH
- Peptides chemistry MeSH
- Proteins chemistry MeSH
- Cross-Linking Reagents chemical synthesis chemistry MeSH
- Serum Albumin, Bovine chemistry MeSH
- Cattle MeSH
- Thymine Nucleotides chemical synthesis chemistry MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.
- MeSH
- DNA-Directed DNA Polymerase metabolism MeSH
- DNA biosynthesis chemistry MeSH
- Inosine Triphosphate analogs & derivatives chemical synthesis metabolism MeSH
- Nucleic Acid Conformation MeSH
- Deoxyribonucleases, Type II Site-Specific metabolism MeSH
- DNA Restriction Enzymes metabolism MeSH
- Substrate Specificity * MeSH
- Vinyl Compounds chemistry MeSH
- Hydrogen Bonding MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.
- MeSH
- Adenosine Triphosphate analogs & derivatives metabolism MeSH
- Bacteriophage T7 enzymology MeSH
- Cytidine Triphosphate analogs & derivatives metabolism MeSH
- DNA-Directed RNA Polymerases metabolism MeSH
- Purine Nucleosides chemistry metabolism MeSH
- Purines chemistry metabolism MeSH
- Pyrimidine Nucleosides chemistry metabolism MeSH
- RNA chemistry metabolism MeSH
- Substrate Specificity MeSH
- Uridine Triphosphate analogs & derivatives metabolism MeSH
- Viral Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
