Cíl studie: Přehled současných poznatků o endokanabinoidech ve vztahu k těhotenství a porodu a jejich potenciálu pro predikci předčasného porodu. Metodika: Přehled publikované literatury k danému tématu použitím databáze PubMed. Výsledky: Kanabinoidní systém hraje roli při vývoji a implantaci blastocysty, embrya, placentaci a u porodu člověka. V lidských tkáních byla identifikována řada zástupců endokanabinoidů, avšak ve vztahu k lidské reprodukci jsou dosud nejvýznamnější anandamid a 2-arachidonoglycerol. Anandamid a jeho následná metabolická přeměna stojí na počátku signální kaskády, která úzce souvisí s délkou těhotenství. Účinky anandamidu závisí na jeho metabolické cestě a aktivitě enzymů, které zajišťují jeho přeměnu. Výsledkem je buď zvýšená tvorba prostaglandinů, nebo prostamidů, s protichůdnými účinky na průběh těhotenství. Užívání exokanabinoidů vč. konopí v období těhotenství ovlivňuje organizmus matky i vývoj plodu na mnoha úrovních a může vést k nepříznivým důsledkům vč. zvýšení rizika předčasného porodu. Závěr: Měření koncentrací anandamidu a stanovení rozmezí poměru prostaglandinů vůči prostamidům pro délku těhotenství by mohlo být užitečným nástrojem při hodnocení rizika předčasného porodu.
Objective: In this paper, we summarize the role of the endocannabinoid system in relation to pregnancy and childbirth and its potential for diagnosis of preterm birth. Methods: Review of articles in peer-reviewed journals using the PubMed database. Results: Endocannabinoid system plays a significant role in embryo development, transport and implantation as well as in placentation. It consists of numerous endogenous ligands; however, in relation to pregnancy there are mainly two studied representatives: anandamide and 2-arachidonoylglycerol. There is increasing evidence, in addition to early pregnancy events, that anandamide plays a regulatory role in pregnancy maintenance and the timing of labour. The activity of anandamide depends on its metabolic pathway and the enzymatic activity that ensures its conversion. Ultimately, changes in anandamide concentration lead to increased production of prostaglandins or prostamides, with inverse effects on pregnancy. The abuse of exogenous cannabinoids in pregnancy has substantial impact on the unborn child in many ways and may result in detrimental effects including preterm birth. Conclusion: Measuring anandamide concentration and the prostaglandin to prostamide ratio could be a useful tool in assessing the risk of preterm birth.
- MeSH
- Endocannabinoids * adverse effects MeSH
- Humans MeSH
- Parturition MeSH
- Pregnancy * MeSH
- Check Tag
- Humans MeSH
- Pregnancy * MeSH
- Female MeSH
- Publication type
- Review MeSH
Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.
- MeSH
- Phosphorylation MeSH
- Caspase 2 chemistry genetics metabolism MeSH
- Protein Conformation * MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Models, Molecular * MeSH
- Protein Multimerization * MeSH
- Mutation MeSH
- Protein Isoforms genetics metabolism MeSH
- Protein Precursors chemistry genetics metabolism MeSH
- 14-3-3 Proteins chemistry genetics metabolism MeSH
- Recombinant Proteins chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.
- MeSH
- DNA chemistry genetics metabolism MeSH
- Transcription, Genetic drug effects MeSH
- Gene Knockdown Techniques MeSH
- HEK293 Cells MeSH
- Small Molecule Libraries chemistry metabolism pharmacology MeSH
- Nucleic Acid Conformation MeSH
- Humans MeSH
- Magnetic Resonance Spectroscopy MeSH
- Models, Molecular MeSH
- Cell Line, Tumor MeSH
- Promoter Regions, Genetic genetics MeSH
- Forkhead Box Protein O3 chemistry genetics metabolism MeSH
- Protein Domains MeSH
- Molecular Docking Simulation MeSH
- Gene Expression Profiling methods MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The catalytic versatility of cytochrome P450 monooxygenases is remarkable. Here, we present mechanistic and structural characterizations of TleB from Streptomyces blastmyceticus and its homolog HinD from Streptoalloteichus hindustanus, which catalyze unusual intramolecular C-N bond formation to generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. In vitro analyses demonstrated that both P450s exhibit promiscuous substrate specificity, and modification of the N13-methyl group resulted in the formation of indole-fused 6/5/6 tricyclic products. Furthermore, X-ray crystal structures in complex with substrates and structure-based mutagenesis revealed the intimate structural details of the enzyme reactions. We propose that the generation of a diradical species is critical for the indolactam formation, and that the intramolecular C(sp2)-H amination is initiated by the abstraction of the N1 indole hydrogen. After indole radical repositioning and subsequent removal of the N13 hydrogen, the coupling of the properly-folded diradical leads to the formation of the C4-N13 bond of indolactam.
Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.
Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).
- MeSH
- Cysteine Endopeptidases chemistry metabolism MeSH
- Phosphorylation MeSH
- Nuclear Localization Signals * MeSH
- Caspase 2 chemistry metabolism MeSH
- Protein Conformation MeSH
- Humans MeSH
- Scattering, Small Angle MeSH
- Models, Molecular MeSH
- 14-3-3 Proteins chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The 14-3-3 proteins, a family of highly conserved scaffolding proteins ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins. Yeast neutral trehalases (Nth), enzymes responsible for the hydrolysis of trehalose to glucose, compared with trehalases from other organisms, possess distinct structure and regulation involving phosphorylation at multiple sites followed by binding to the 14-3-3 protein. Here we report the crystal structures of yeast Nth1 and its complex with Bmh1 (yeast 14-3-3 isoform), which, together with mutational and fluorescence studies, indicate that the binding of Nth1 by 14-3-3 triggers Nth1's activity by enabling the proper 3D configuration of Nth1's catalytic and calcium-binding domains relative to each other, thus stabilizing the flexible part of the active site required for catalysis. The presented structure of the Bmh1:Nth1 complex highlights the ability of 14-3-3 to modulate the structure of a multidomain binding partner and to function as an allosteric effector. Furthermore, comparison of the Bmh1:Nth1 complex structure with those of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock protein beta-6 complexes revealed similarities in the 3D structures of bound partner proteins, suggesting the highly conserved nature of 14-3-3 affects the structures of many client proteins.
- MeSH
- Arylalkylamine N-Acetyltransferase metabolism MeSH
- Databases, Chemical * MeSH
- Phosphorylation MeSH
- Glucose metabolism MeSH
- Catalytic Domain MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray MeSH
- Models, Molecular MeSH
- Protein Domains MeSH
- 14-3-3 Proteins genetics metabolism MeSH
- Heat-Shock Proteins chemistry metabolism MeSH
- Saccharomyces cerevisiae Proteins chemistry metabolism MeSH
- Saccharomyces cerevisiae enzymology genetics metabolism MeSH
- Trehalase chemistry metabolism MeSH
- Trehalose metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Procaspase-2 phosphorylation at several residues prevents its activation and blocks apoptosis. This process involves procaspase-2 phosphorylation at S164 and its binding to the scaffolding protein 14-3-3. However, bioinformatics analysis has suggested that a second phosphoserine-containing motif may also be required for 14-3-3 binding. In this study, we show that human procaspase-2 interaction with 14-3-3 is governed by phosphorylation at both S139 and S164. Using biochemical and biophysical approaches, we show that doubly phosphorylated procaspase-2 and 14-3-3 form an equimolar complex with a dissociation constant in the nanomolar range. Furthermore, our data indicate that other regions of procaspase-2, in addition to phosphorylation motifs, may be involved in the interaction with 14-3-3.
- MeSH
- Phosphorylation MeSH
- Caspase 2 chemistry metabolism MeSH
- Humans MeSH
- Protein Domains MeSH
- 14-3-3 Proteins metabolism MeSH
- Recombinant Proteins chemistry metabolism MeSH
- Amino Acid Sequence MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
