multifunctional structures
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Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.
- MeSH
- endodeoxyribonukleasy chemie genetika metabolismus MeSH
- fosfáty metabolismus MeSH
- krystalizace MeSH
- molekulární sekvence - údaje MeSH
- multienzymové komplexy chemie genetika metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Solanum lycopersicum enzymologie genetika MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.
- MeSH
- deoxyribonukleasy chemie metabolismus MeSH
- fosfolipasy chemie metabolismus MeSH
- katalytická doména MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- multienzymové komplexy chemie metabolismus MeSH
- myši MeSH
- rostlinné proteiny chemie metabolismus MeSH
- Solanum lycopersicum enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (Aβ), glycogen synthase kinase 3β (GSK-3β) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3β (IC50 = 24.36 ± 0.01 μM) and Aβ42 self-aggregation (IC50 = 9.0 ± 1.4 μM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 ± 0.5 · 105 M-1s-1) even in phosphate buffer at pH 7.4 (kinh = 3.2 ± 0.5 · 105 M-1s-1). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 μM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- cinnamáty chemická syntéza chemie farmakologie MeSH
- kinasa glykogensynthasy 3beta antagonisté a inhibitory metabolismus MeSH
- molekulární struktura MeSH
- scavengery volných radikálů chemická syntéza chemie farmakologie MeSH
- stereoizomerie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nucleolin is the major nucleolar protein of animal, plant and yeast proliferating cells. It is enriched in the most soluble nuclear or nucleolar protein extract, containing ribonucleoproteins, from which it has been purified. It has a tripartite structure in which each domain accounts for different functions. Despite its multifunctionality, the best characterized role of nucleolin is in the primary cleavage of pre-rRNA, an early step of ribosome biogenesis. In the nucleolus of proliferating cells, nucleolin is mostly located in the dense fibrillar component, following a vectorial pattern, from the periphery of fibrillar centers outwards. This pattern is lost in quiescent cells in which nucleolin is present in low levels. Nucleolin is the most phosphorylated protein of the soluble nuclear extract. It is phosphorylated by casein kinase II and CDKA, and phosphorylation is closely associated with the role of nucleolin in proliferating cells. During mitosis, nucleolin is transported from the mother to the daughter cell nucleolus in the form of processing particles, together with pre-rRNA precursors and other nucleolar proteins. It forms part of prenucleolar bodies and plays a role in nucleologenesis. Recent studies on the nucleolin function, carried out on samples with inactivated nucleolin genes (siRNA downregulated or mutants) have evidenced that nucleolin is absolutely essential for cell proliferation, for the organization of the nucleolus and for transcription and processing of pre-rRNA. In plants, nucleolin controls the auxin responsiveness, thus being involved in the regulation of plant development.
- MeSH
- biogeneze organel MeSH
- buněčný cyklus fyziologie genetika MeSH
- fosfoproteiny biosyntéza genetika chemie MeSH
- fosforylace fyziologie genetika MeSH
- jaderné proteiny biosyntéza genetika chemie MeSH
- klinické laboratorní techniky využití MeSH
- kvasinky MeSH
- kyseliny indoloctové MeSH
- prekurzory RNA biosyntéza genetika chemie MeSH
- proliferace buněk MeSH
- proteiny vázající RNA biosyntéza genetika chemie MeSH
- ribonukleoproteiny malé jadérkové biosyntéza MeSH
- ribozomy genetika chemie MeSH
- rostliny MeSH
- statistika jako téma MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).
- MeSH
- Candida metabolismus MeSH
- desaturasy mastných kyselin metabolismus MeSH
- fylogeneze MeSH
- kyselina linolová metabolismus MeSH
- kyseliny ricinolejové metabolismus MeSH
- molekulární sekvence - údaje MeSH
- nenasycené mastné kyseliny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Multifunctional two-photon laser scanning microscopy provides attractive advantages over conventional two-photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. RESULTS: These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two-photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. CONCLUSIONS: With the multifunctional two-photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.
- MeSH
- chloroplasty chemie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- listy rostlin chemie ultrastruktura MeSH
- mikroskopie fluorescenční multifotonová metody MeSH
- počítačové zpracování obrazu metody MeSH
- Selaginellaceae chemie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Looking for an effective anti-Alzheimer's agent is very challenging; however, a multifunctional ligand strategy may be a promising solution for the treatment of this complex disease. We herein present the design, synthesis and biological evaluation of novel hydroxyethylamine derivatives displaying unique, multiple properties that have not been previously reported. The original mechanism of action combines inhibitory activity against disease-modifying targets: β-secretase enzyme (BACE1) and amyloid β (Aβ) aggregation, along with an effect on targets associated with symptom relief - inhibition of butyrylcholinesterase (BuChE) and γ-aminobutyric acid transporters (GATs). Among the obtained molecules, compound 36 exhibited the most balanced and broad activity profile (eeAChE IC50 = 2.86 μM; eqBuChE IC50 = 60 nM; hBuChE IC50 = 20 nM; hBACE1 IC50 = 5.9 μM; inhibition of Aβ aggregation = 57.9% at 10 μM; mGAT1 IC50 = 10.96 μM; and mGAT2 IC50 = 19.05 μM). Moreover, we also identified 31 as the most potent mGAT4 and hGAT3 inhibitor (IC50 = 5.01 μM and IC50 = 2.95 μM, respectively), with high selectivity over other subtypes. Compounds 36 and 31 represent new anti-Alzheimer agents that can ameliorate cognitive decline and modify the progress of disease.
- MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- molekulární struktura MeSH
- neuroprotektivní látky chemická syntéza chemie farmakologie MeSH
- objevování léků * MeSH
- proteiny přenášející GABA přes plazmatickou membránu metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.
Onion (Allium cepa L.) contains various bioactive organosulfur compounds, including recently identified species with a 3,4-dimethylthiolane ring structure. In this study, the biological activities of 24 onion-derived thiolane samples were comprehensively evaluated, focusing on antioxidant, anti-inflammatory, antidiabetic, and antimicrobial potential. Tested thiolanes showed significant antioxidant activity by reducing intracellular reactive oxygen species (ROS) levels in THP1-BlueTM NF-κB cells; however, some tested derivatives exhibited mild pro-oxidant effects. In assays targeting metabolic regulation, several compounds showed partial activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway, particularly allithiolane B isomers, achieving 22-26% of the maximal response induced by the known full agonist rosiglitazone, suggesting potential as PPARγ modulators. Anti-inflammatory assessments revealed minimal direct inhibition of NF-κB signaling, except for onionin A, which modestly attenuated NF-κB activity. Cytotoxic evaluation indicated safety at physiological concentrations (up to 10 μM), though some thiolanes significantly reduced cell viability at higher concentrations (50 μM), highlighting their potential for selective cytostatic applications. Antimicrobial screening indicated limited activity against bacteria and fungi at tested concentrations, except for moderate antifungal effectiveness against Candida albicans shown by allithiolane D and cepathiolactols F. Overall, these results emphasise that onion thiolanes are multifunctional molecules capable of modulating oxidative stress, metabolic regulation and inflammation, supporting their potential use in nutraceutical strategies aimed at chronic disease prevention and management.
- MeSH
- antiflogistika farmakologie chemie MeSH
- antiinfekční látky farmakologie chemie MeSH
- antioxidancia farmakologie chemie MeSH
- buněčné linie MeSH
- česneky * chemie MeSH
- hypoglykemika farmakologie chemie MeSH
- lidé MeSH
- NF-kappa B metabolismus genetika MeSH
- PPAR gama metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- rostlinné extrakty * farmakologie chemie MeSH
- sulfhydrylové sloučeniny * farmakologie chemie MeSH
- viabilita buněk účinky léků MeSH
- zelenina * chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.
- Publikační typ
- časopisecké články MeSH
