RESUMEN
The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.
Asunto(s)
Adenilosuccinato Sintasa , Helicobacter pylori , Histidina , Helicobacter pylori/enzimología , Histidina/metabolismo , Histidina/química , Adenilosuccinato Sintasa/metabolismo , Adenilosuccinato Sintasa/química , Adenilosuccinato Sintasa/genética , Cinética , Dicroismo Circular , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Difracción de Rayos XRESUMEN
Auxin amino acid conjugates are considered to be storage forms of auxins. Previous research has shown that indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala) and indole-3-butyryl-L-alanine (IBA-Ala) affect the root growth of Brassica rapa seedlings. To elucidate the potential mechanism of action of the conjugates, we treated B. rapa seedlings with 0.01 mM IAA-, IPA- and IBA-Ala and investigated their effects on the auxin metabolome and transcriptome. IBA-Ala and IPA-Ala caused a significant inhibition of root growth and a decrease in free IAA compared to the control and IAA-Ala treatments. The identification of free auxins IBA and IPA after feeding experiments with IBA-Ala and IPA-Ala, respectively, confirms their hydrolysis in vivo and indicates active auxins responsible for a stronger inhibition of root growth. IBA-Ala caused the induction of most DEGs (807) compared to IPA-Ala (417) and IAA-Ala (371). All treatments caused similar trends in transcription profile changes when compared to control treatments. The majority of auxin-related DEGs were found after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, which is consistent with the apparent root morphology. In addition to most YUC genes, which showed a tendency to be downregulated, transcripts of auxin-related DEGs that were identified (UGT74E2, GH3.2, SAUR, IAA2, etc.) were more highly expressed after all treatments. Our results are consistent with the hypothesis that the hydrolysis of conjugates and the release of free auxins are responsible for the effects of conjugate treatments. In conclusion, free auxins released by the hydrolysis of all auxin conjugates applied affect gene regulation, auxin homeostasis and ultimately root growth inhibition.
Asunto(s)
Brassica rapa , Gastrópodos , Animales , Ácidos Indolacéticos/farmacología , Brassica rapa/genética , Transcriptoma , Indoles , Alanina , Plantones/genéticaRESUMEN
Newly designed and synthesized diarylethene (DAE) derivatives with aliphatic amine sidearms and one with two pyrenes, revealed excellent photo-switching property of central DAE core in MeOH and water. The only exception was bis-pyrene analogue, its DAE core very readily photochemically closed, but reversible opening completely hampered by aromatic stacking interaction of pyrene(s) with cyclic DAE. In this process, pyrene fluorescence showed to be a reliable monitoring method, an open form characterized by strong emission at 480 nm (typical for pyrene-aggregate), while closed form emitted weakly at 400 nm (typical for pyrene-DAE quenching). Only open DAE-bis-pyrene form interacted measurably with ds-DNA/RNA by flexible insertion in polynucleotide grooves, while self-stacked closed form did not bind to DNA/RNA. For the same steric reasons, flexible open DAE-bis-pyrene form was bound to at least three different binding sites at bovine serum albumin (BSA), while rigid, self-stacked closed form interacted dominantly with only one BSA site. Preliminary screening of antiproliferative activity against human lung carcinoma cell line A549 revealed that all DAE-derivatives are non-toxic. However, bis-pyrene analogue efficiently entered cells and located in the cytoplasm, whereby irradiation by light (315-400 nm) resulted in a strong, photo-induced cytotoxic effect, typical for pyrene-related singlet oxygen species production.
Asunto(s)
ADN/química , Luz , Conformación Molecular , Pirenos/química , Albúmina Sérica Bovina/química , Células A549 , Animales , Bovinos , Supervivencia Celular , Electrones , Humanos , Desnaturalización de Ácido Nucleico/efectos de la radiación , Procesos Fotoquímicos , ARN/química , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptófano/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Dominio Catalítico/genética , Dicroismo Circular , ADN/química , ADN/genética , ADN Helicasas/química , ADN Helicasas/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edición Génica/métodos , Humanos , Mutación Missense , Conformación Proteica , Homología de Secuencia de Aminoácido , Temperatura , Triptófano/química , Triptófano/genéticaRESUMEN
Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10-2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10-5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105-106 M-1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M-1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.
Asunto(s)
Antineoplásicos/farmacología , Indolquinonas/síntesis química , Naftalimidas/química , Naftoles/química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Transferencia Resonante de Energía de Fluorescencia , Humanos , Indolquinonas/química , Indolquinonas/farmacología , Células MCF-7 , Estructura Molecular , Procesos Fotoquímicos , Teoría CuánticaRESUMEN
Photochemical reactivity of pentacycloundecane (PCU) and adamantane diazirines was investigated by preparative irradiation in different solvents, laser flash photolysis (LFP) and quantum chemical computations. In addition, formation of inclusion complexes for diazirines with cucurbit[7]uril, ß- and γ-cyclodextrin (ß- and γ-CD) was investigated by 1H NMR spectroscopy, isothermal microcalorimetry and circular dichroism spectroscopy, followed by the investigation of photochemical reactivity of the formed complexes. Diazirines undergo efficient photochemical elimination of nitrogen (ΦR > 0.5) and deliver the corresponding singlet carbenes. Singlet carbenes react in intra- and intermolecular reactions and we found a rare singlet carbene pathway in CH3OH involving protonation and formation of a carbocation, detected due to the specific rearrangement of the pentacycloundecane skeleton. Singlet diazirines undergo intersystem crossing and deliver triplet carbenes that react with oxygen to form ketones which were isolated after irradiation. Our main finding is that the formation of diazirine inclusion complexes with ß-CD and γ-CD changes the relative ratio of singlet vs. triplet pathways, with singlet carbene products being dominant from the chemistry of the irradiated complexes. Our combined theoretical and experimental studies provide new insights into the supramolecular control of carbene reactivity which has possible applications for the control of product distribution by solvent effects and the choice of constrained media.
RESUMEN
We demonstrate a proof of principle for a new approach in the development of a drug delivery system. A positively charged prodrug (phenol) can form a stable inclusion complex with CB[7], which enables more efficient delivery of the prodrug. After photochemical transformation (photoactivation) inside the complex, an active drug quinone methide (QM) is formed and released from the complex, since it is a neutral molecule and forms a less stable complex with CB[7].
Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Preparaciones de Acción Retardada/química , Imidazoles/química , Indolquinonas/administración & dosificación , Fenol/administración & dosificación , Profármacos/administración & dosificación , Sistemas de Liberación de Medicamentos , Indolquinonas/química , Fenol/química , Procesos Fotoquímicos , Profármacos/químicaRESUMEN
This work reports two new peptide-based fluorescence probes (1 and 2) for the detection of ds-DNA at physiological pH. Probes 1 and 2 contain a fluorophore, either amino-naphthalimide or diethyl-aminocoumarin, respectively, and two identical peptide arms each equipped with a guanidiniocarbonylpyrrole (GCP) anion-binding motif. These probes show "switch-on" fluorescence response upon binding to ds-DNA, whereby they can differentiate between various types of polynucleotides. For instance, they exhibit more pronounced fluorescence response for AT-rich polynucleotides than GC-rich polynucleotides, and both give only negligible response to ds-RNA. The fluorimetric response of 1 is proportional to the AT-basepair content in DNA, whereas the fluorescence of 2 is sensitive to the secondary structure of the polynucleotide. Fluorescence experiments, thermal melting experiments and circular dichroism studies suggest that 1 interacts with ds-DNA in a combined intercalation and minor groove binding, whereas 2 interacts mainly with the outer surface of DNA/RNA. As 1 and 2 have a very low cytotoxicity, 1 can be applied for the imaging of nuclear DNA in cells.
Asunto(s)
ADN/análisis , Colorantes Fluorescentes/química , Péptidos/química , Células A549 , Animales , Aniones/química , Bovinos , Dicroismo Circular , Cumarinas/química , ADN/química , Colorantes Fluorescentes/síntesis química , Humanos , Microscopía Confocal , Naftalimidas/química , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
An arginine mimetic, featuring a guanidiniocarbonypyrrol as artificial anion binding site (GCP), was introduced into short peptides to study their binding and aggregation with double stranded DNA and RNA. While the incorporation of this GCP modification did not significantly change the overall binding affinity for DNA/RNA, their interactions were more sensitive with respect to the type of polynucleotides. Peptide 4, with four GCP modifications, exhibited amazing fluorescent selectivity for AU- and (to a lesser extent) AT- sequences. GC-containing DNA showed much lower response. Additionally, exclusively tri-GCP modified peptide 3 showed intriguing exciton-coupled bisignate ICD bands for all studied DNA/RNA which suggested that a well-defined GCP-dimer bound into DNA/RNA groove..
Asunto(s)
Arginina/química , ADN/química , Imitación Molecular , Péptidos/química , ARN Bicatenario/química , Cromatografía Líquida de Alta Presión , Desnaturalización de Ácido Nucleico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Análisis Espectral/métodosRESUMEN
G-Quadruplex (G4) structures of a human telomeric 24-mer (5'-TTAGGGTTAGGGTTAGGGTTAGGG-3') sequence (Tel24) stabilized by sodium and potassium ions have been assessed using surface-enhanced Raman scattering (SERS) spectroscopy. The distinctive SERS spectra of Tel24 in the presence of 100 mM Na+ and 100 mM K+ were obtained and the SERS bands characteristic of the antiparallel basket-type and the mixed hybrid (3+1) structures, respectively, were identified and assigned. The influence of the SERS - active substrate on the scattering enhancement was studied using citrate- and chloride-covered silver nanoparticles, in the absence and presence of the aggregating agent (0.1 M Na2SO4 and 0.1 M K2SO4). The highly reproducible SERS spectra of Tel24 obtained in various SERS active media indicated the same adsorption mechanism of the cation - stabilized G-quadruplexes onto the metal surface, regardless of the silver colloid. The remarkable resemblance between the circular dichroism (CD) spectra of the Tel24 structures with and without the colloid confirmed that interaction with the enhancing silver surface did not affect the stability of the formed G4 structures. The presented study pointed to a great potential of the SERS spectroscopy for the sensitive structural analysis of various G4 topologies. Graphical Abstract SERS spectroscopy allowed identification of Na+ stabilized antiparallel basket-type and K+ stabilized hybrid (3+1) structures of the same 24-mer human telomeric sequence.
Asunto(s)
G-Cuádruplex , Espectrometría Raman/métodos , Telómero , Adsorción , Dicroismo Circular , HumanosRESUMEN
Several new adamantyl and homoadamantyl-substituted [Formula: see text]-hydroxybutyric acids, 2-[2-(1-adamantyl)ethyl]-3-hydroxybutyric acid (2), 2-(3-homoadamantyl)-3-hydroxybutyric acid (3), and 2-(1-homoadamantyl)-3-hydroxybutyric acid (4), analogues of the 2-(1-adamantyl)-3-hydroxybutyric acid (1), have been prepared as mixtures of diastereoisomers using selective reduction of corresponding [Formula: see text]-keto esters or aldol condensation of the corresponding carboxylic acid and acetaldehyde. The rearrangement of adamantylmethyl and 3-homoadamantyl groups provided entry to both 3-homoadamantyl and 1-homoadamantyl-substituted hydroxy acids 3 and 4, respectively. The relative configurations of diastereoisomers 3 and 4 have been determined by NMR spectroscopy comparing the values of coupling constants. Adamantyl-substituted [Formula: see text]-hydroxybutyric acid 2 has also been prepared in enantiomerically pure form by Evan's asymmetric synthesis and the absolute configuration has been determined by X-ray crystallography. Contrary to the long-chain acid 2, the attempt to prepare short-chain hydroxy acids 1 and 4 by the same method failed indicating pronounced sensitivity of the used method to the vicinity of the bulky cage group.
Asunto(s)
Ácido 3-Hidroxibutírico/síntesis química , Ácido 3-Hidroxibutírico/química , Técnicas Químicas Combinatorias , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
The actual cumulative mass of released quercetin from nanoparticles within the dialysis membrane was determined under the influence of external stationary and alternating magnetic fields. We have shown that the control of the release kinetics of quercetin from MNPs, i.e., the distribution of quercetin between the nanoparticles and the suspension within the membrane, can be tuned by the simple combination of stationary and alternating magnetic fields. Under non-sink conditions, the proportion of quercetin in the suspension inside the membrane is increased toward the nanoparticles, resulting in the increased release of quercetin. The results obtained could be applied to the release of insoluble flavonoids in aqueous suspensions in general.
RESUMEN
ADP-ribosylation is an ancient, highly conserved, and reversible covalent modification critical for a variety of endogenous processes in both prokaryotes and eukaryotes. ADP-ribosylation targets proteins, nucleic acids, and small molecules (including antibiotics). ADP-ribosylation signalling involves enzymes that add ADP-ribose to the target molecule, the (ADP-ribosyl)transferases; and those that remove it, the (ADP-ribosyl)hydrolases. Recently, the toxin/antitoxin pair DarT/DarG composed of a DNA ADP-ribosylating toxin, DarT, and (ADP-ribosyl)hydrolase antitoxin, DarG, was described. DarT modifies thymidine in single-stranded DNA in a sequence-specific manner while DarG reverses this modification, thereby rescuing cells from DarT toxicity. We studied the DarG homologue SCO6735 which is highly conserved in all Streptomyces species and known to be associated with antibiotic production in the bacterium S. coelicolor. SCO6735 shares a high structural similarity with the bacterial DarG and human TARG1. Like DarG and TARG1, SCO6735 can also readily reverse thymidine-linked ADP-ribosylation catalysed by DarT in vitro and in cells. SCO6735 active site analysis including molecular dynamic simulations of its complex with ADP-ribosylated thymidine suggests a novel catalytic mechanism of DNA-(ADP-ribose) hydrolysis. Moreover, a comparison of SCO6735 structure with ALC1-like homologues revealed an evolutionarily conserved feature characteristic for this subclass of macrodomain hydrolases.
RESUMEN
A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.
Asunto(s)
Adamantano/análogos & derivados , Adamantano/síntesis química , Aminoácidos/síntesis química , Antineoplásicos/síntesis química , Encefalina Metionina/análogos & derivados , Encefalina Metionina/síntesis química , Oligopéptidos/síntesis química , Adamantano/química , Adamantano/farmacología , Aminoácidos/química , Aminoácidos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Encefalina Metionina/química , Encefalina Metionina/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Oligopéptidos/química , Oligopéptidos/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
1,3-Diaryltriazenes (1) were let to react with [RuCl2(p-cymene)]2 in the presence of trimethylamine to give neutral 1,3-diaryltriazenido(p-cymene)ruthenium(II) complexes, [RuCl(p-cymene)(ArNNNAr)] (2). The molecular composition of the products 2 was confirmed by NMR spectroscopy and mass spectrometry. The structures of the selected complexes were confirmed by a single crystal X-ray analysis. All triazenido-ruthenium complexes were highly cytotoxic against human cervical carcinoma HeLa cells with IC50 below 6µM, as determined by a spectrophotometric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method. The most active was [RuCl(p-cymene)(ArNNNAr)] (Ar=4-Cl-3-(CF3)-C6H3) (2g) with IC50 of 0.103±0.006µM. In comparison with the data for the non-coordinated triazenes 1, the triazenido-ruthenium complexes 2 exhibited up to 560-times higher activity. Three selected complexes were highly cytotoxic also against several tumor cell lines: laryngeal carcinoma HEp-2 cells and their drug-resistant HEp-2 subline (7T), colorectal carcinoma HCT-116 cells, lung adenocarcinoma H460 cells, and mammary carcinoma MDA-MB-435 cells. The compounds 2g and [RuCl(p-cymene)(ArNNNAr)] (Ar=4-I-C6H4) (2j) were similarly cytotoxic against parental and drug-resistant cells. Time and dose dependent accumulation of the cells in the S phase of the cell cycle was induced by the compound 2g, triggering apoptosis. Our preliminary results indicate triazenido-ruthenium complexes as promising anticancer drug candidates.