Vitamin D je skupina steroidních hormonů. Většina v těle vzniká za pomoci UV záření ze slunce, ale je obsažen v různých potravinách, jako jsou oleje z mořských ryb apod. V těle je postupně hydroxylován na účinný metabolit v játrech a ledvinách. V krvi je transportován bílkovinou VDBP (vitamin D binding protein). Váže se v jádře buňky na receptor VDR (vitamin D Rreceptor). Na koncentraci vitaminu D má vliv mnoho faktorů jako zeměpisná poloha, sezóna (délka slunečního svitu), pigmentace kůže i množství tukové a svalové tkáně. Jeho nejznámější funkcí je regulace kalcio-fosfátového metabolizmu, avšak podílí se rovněž na regulaci buněčného cyklu, indukci apoptózy a také hraje roli v regulaci imunitního systému. Obecně lze říci, že jeho působení vede spíše k imunotoleranci. Nedostatek vitaminu D se v populaci projevuje stále častěji, dnes jím trpí až téměř 50 % evropské populace. Deficience se spojuje s vyšší agresivitou nádorů vč. Nehodgkinových lymfomů a je prokázáno, že pacienti s vyššími hladinami vitaminu D vykazují lepší celkové přežití i dobu do progrese. Nabízí se tedy otázka, zda by suplementace vitaminem D mohla příznivě ovlivnit prognózu pacienta s lymfomy. Výsledky publikovaných studií jsou v tomto ohledu dosud rozporuplné. Navzdory ne zcela jednoznačným výsledkům se uvádí, že suplementace by měla být zvážena u pacientů s insuficientními hladinami vitaminu D.
Vitamin D is a group of steroid hormones, produced with the help of UV radiation of the sun in the skin. It is also contained in various foods such as marine fish oils etc. In the body, it is subsequently transformed into its active form in the liver and kidneys. In the blood, it is transported by the VDBP (vitamin D binding protein). In the cell nucleus, it is bound to the VDR receptor (vitamin D receptor). The concentration of vitamin D in plasma is influenced by many factors: geographical latitude, season (length of sunshine), skin pigmentation, amount of fat, and muscle tissue. The best-known function of vitamin D is the regulation of calcium-phosphate metabolism, but it is involved in many processes such as the regulation of the cell cycle and the induction of apoptosis. It plays a role in the regulation of the immune system as well. Its immunomodulatory action is required for adequate anti-infectious and anti-tumoral immune response. It prevents an exaggerated inflammatory reaction and leads to immunotolerance. Deficiency has become more common in our population, affecting up to 50% of Europeans. Deficiency is also associated with a higher aggressiveness of tumours, including non-Hodgkin lymphomas. It has been shown that higher levels of vitamin D are associated with better overall survival and time to progression. The question is, whether vitamin D supplementation could impact and improve prognosis. Despite the ambiguous results of published studies, vitamin D supplementation should be considered in patients with diagnosed deficiency.
- MeSH
- Humans MeSH
- Lymphoma * etiology therapy MeSH
- Vitamin D Deficiency complications MeSH
- Prognosis MeSH
- Retrospective Studies MeSH
- Vitamin D * immunology metabolism therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
Retinitis pigmentosa (RP) is a hereditary disorder caused by mutations in more than 70 different genes including those that encode proteins important for pre-mRNA splicing. Most RP-associated mutations in splicing factors reduce either their expression, stability or incorporation into functional splicing complexes. However, we have previously shown that two RP mutations in PRPF8 (F2314L and Y2334N) and two in SNRNP200 (S1087L and R1090L) behaved differently, and it was still unclear how these mutations affect the functions of both proteins. To investigate this in the context of functional spliceosomes, we used iCLIP in HeLa and retinal pigment epithelial (RPE) cells. We found that both mutations in the RNA helicase SNRNP200 change its interaction with U4 and U6 snRNAs. The significantly broader binding profile of mutated SNRNP200 within the U4 region upstream of the U4/U6 stem I strongly suggests that its activity to unwind snRNAs is impaired. This was confirmed by FRAP measurements and helicase activity assays comparing mutant and WT protein. The RP variants of PRPF8 did not affect snRNAs, but showed a reduced binding to pre-mRNAs, which resulted in the slower splicing of introns and altered expression of hundreds of genes in RPE cells. This suggests that changes in the expression and splicing of specific genes are the main driver of retinal degeneration in PRPF8-linked RP.
- MeSH
- HeLa Cells MeSH
- Humans MeSH
- Ribonucleoprotein, U4-U6 Small Nuclear metabolism genetics MeSH
- Mutation * MeSH
- Eye Proteins genetics metabolism MeSH
- RNA Precursors * metabolism genetics MeSH
- RNA-Binding Proteins metabolism genetics MeSH
- Retinal Pigment Epithelium metabolism pathology MeSH
- Retinitis Pigmentosa * genetics metabolism pathology MeSH
- Ribonucleoproteins, Small Nuclear metabolism genetics MeSH
- RNA, Small Nuclear genetics metabolism MeSH
- RNA Splicing * genetics MeSH
- Spliceosomes metabolism genetics MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The vertebrate visual cycle hinges on enzymatically converting all-trans-retinol (at-ROL) into 11-cis-retinal (11c-RAL), the chromophore that binds to opsins in photoreceptors, forming light-responsive pigments. When struck by a photon, these pigments activate the phototransduction pathway and initiate the process of vision. The enzymatic isomerization of at-ROL, crucial for restoring the visual pigments and preparing them to receive new light stimuli, relies on various enzymes found in both the photoreceptors and retinal pigment epithelium cells. To function effectively, retinoids must shuttle between these two cell types. Retinol-binding protein 3 (RBP3), located in the interphotoreceptor matrix, probably plays a pivotal role in this transport mechanism. Comprised of four retinoid-binding modules, RBP3 also binds fatty acids, potentially aiding retinal function by facilitating the loading and unloading of different retinoids at specific cell types thereby directing the cycle. In this study, we present a 3.67 Å cryoEM structure of porcine RBP3, along with molecular docking analysis and corroborative in-solution small-angle X-ray scattering data for titration of RBP3 with relevant ligands, that also give insights on RBP3 conformational adaptability.
- MeSH
- X-Ray Diffraction MeSH
- Cryoelectron Microscopy methods MeSH
- Protein Conformation MeSH
- Scattering, Small Angle * MeSH
- Models, Molecular MeSH
- Eye Proteins MeSH
- Swine MeSH
- Retinol-Binding Proteins * chemistry metabolism MeSH
- Molecular Docking Simulation MeSH
- Protein Binding MeSH
- Vitamin A metabolism chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
UNLABELLED: Staphylococcus aureus is a lethal pathogen that can cause various bacterial infections. This study targets the CrtM enzyme of S. aureus, which is crucial for synthesizing golden carotenoid pigment: staphyloxanthin, which provides anti-oxidant activity to this bacterium for combating antimicrobial resistance inside the host cell. The present investigation quests for human SQS inhibitors against the CrtM enzyme by employing structure-based drug design approaches including induced fit docking (IFD), molecular dynamic (MD) simulations, and binding free energy calculations. Depending upon the docking scores, two compounds, lapaquistat acetate and squalestatin analog 20, were identified as the lead molecules exhibit higher affinity toward the CrtM enzyme. These docked complexes were further subjected to 100 ns MD simulation and several thermodynamics parameters were analyzed. Further, the binding free energies (ΔG) were calculated for each simulated protein-ligand complex to study the stability of molecular contacts using the MM-GBSA approach. Pre-ADMET analysis was conducted for systematic evaluation of physicochemical and medicinal chemistry properties of these compounds. The above study suggested that lapaquistat acetate and squalestatin analog 20 can be selected as potential lead candidates with promising binding affinity for the S. aureus CrtM enzyme. This study might provide insights into the discovery of potential drug candidates for S. aureus with a high therapeutic index. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-023-03862-y.
- Publication type
- Journal Article MeSH
Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic bacterium that predominantly infects infants in developing countries. EPEC forms attaching and effacing (A/E) lesions on the apical surface of the small intestine, leading to diarrhea. The locus of enterocyte effacement (LEE) is both necessary and sufficient for A/E lesion morphogenesis by EPEC. Gene expression from this virulence determinant is controlled by an elaborate regulatory web that extends beyond protein-based transcriptional regulators and includes small regulatory RNA (sRNA) that exert their effects posttranscriptionally. To date, only 4 Hfq-dependent sRNAs-MgrR, RyhB, McaS, and Spot42-have been identified that affect the LEE of EPEC by diverse mechanisms and elicit varying regulatory outcomes. In this study, we demonstrate that the paralogous Hfq-dependent sRNAs OmrA and OmrB globally silence the LEE to diminish the ability of EPEC to form A/E lesions. Interestingly, OmrA and OmrB do not appear to directly target a LEE-encoded gene; rather, they repress transcription from the LEE1 promoter indirectly, by means of an as-yet-unidentified transcriptional factor that binds within 200 base pairs upstream of the transcription start site to reduce the expression of the LEE master regulator Ler, which, in turn, leads to reduced morphogenesis of A/E lesions. Additionally, OmrA and OmrB also repress motility in EPEC by targeting the 5' UTR of the flagellar master regulator, flhD.
- MeSH
- Coloring Agents MeSH
- Enteropathogenic Escherichia coli * MeSH
- Infant MeSH
- Humans MeSH
- Promoter Regions, Genetic MeSH
- Diarrhea MeSH
- Transcription Factors MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.
- MeSH
- Coloring Agents MeSH
- Lysine MeSH
- Peptides MeSH
- Sirtuin 1 metabolism MeSH
- Sirtuin 2 metabolism MeSH
- Sirtuin 3 * metabolism MeSH
- Sirtuins * metabolism MeSH
- Publication type
- Journal Article MeSH
Spitz tumors are melanocytic neoplasms characterized by specific, mutually exclusive driver molecular events, namely genomic rearrangements involving the threonine kinase BRAF and the tyrosine kinase receptors ALK , NTRK1 , NTRK2 , NTRK3 , MET , RET , ROS1 , and MAP3K8 or less commonly, mutations in HRAS or MAP2K1 . We hereby report 5 Spitz tumors with a SQSTM1::NTRK2 fusion. All patients were woman with the ages at diagnosis ranging from 30 to 50 years. Locations included the lower extremity (n = 3), forearm, and back (one each). All the neoplasms were superficial melanocytic proliferation with a flat to dome-shaped silhouette, in which junctional spindled and polygonal dendritic melanocytes were mainly arranged as horizontal nests associated with conspicuous lentiginous involvement of the follicular epithelium. Only one case showed heavily pigmented, vertically oriented melanocytic nests resembling Reed nevus. A superficial intradermal component observed in 2 cases appeared as small nests with a back-to-back configuration. In all lesions, next-generation sequencing analysis identified a SQSTM1::NTRK2 fusion. A single case studied with fluorescence in situ hybridization for copy number changes in melanoma-related genes proved negative. No further molecular alterations were detected, including TERT-p hotspot mutations.
- MeSH
- Adult MeSH
- Nevus, Epithelioid and Spindle Cell * genetics MeSH
- In Situ Hybridization, Fluorescence MeSH
- Middle Aged MeSH
- Humans MeSH
- Skin Neoplasms * pathology MeSH
- Proto-Oncogene Proteins genetics MeSH
- Sequestosome-1 Protein genetics MeSH
- Receptor Protein-Tyrosine Kinases genetics MeSH
- Protein-Tyrosine Kinases genetics MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 μm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 μm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.
- MeSH
- Bestrophins metabolism MeSH
- Retinal Degeneration * metabolism MeSH
- Cells, Cultured MeSH
- Nanofibers * chemistry MeSH
- Polyesters metabolism MeSH
- Swine MeSH
- Retinal Pigment Epithelium metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Tyrosinase, exquisitely catalyzes the phenolic compounds into brown or black pigment, inhibition is used as a treatment for dermatological or neurodegenerative disorders. Natural products, such as cyanidin-3-O-glucoside and (-/+)-catechin, are considered safe and non-toxic food additives in tyrosinase inhibition but their ambiguous inhibitory mechanism against tyrosinase is still elusive. Thus, we presented the mechanistic insights into tyrosinase with cyanidin-3-O-glucoside and (-/+)-catechin using computational simulations and in vitro assessment. Initial molecular docking results predicted ideal docked poses (- 9.346 to - 5.795 kcal/mol) for tyrosinase with selected flavonoids. Furthermore, 100 ns molecular dynamics simulations and post-simulation analysis of docked poses established their stability and oxidation of flavonoids as substrate by tyrosinase. Particularly, metal chelation via catechol group linked with the free 3-OH group on the unconjugated dihydropyran heterocycle chain was elucidated to contribute to tyrosinase inhibition by (-/+)-catechin against cyanidin-3-O-glucoside. Also, predicted binding free energy using molecular mechanics/generalized Born surface area for each docked pose was consistent with in vitro enzyme inhibition for both mushroom and murine tyrosinases. Conclusively, (-/+)-catechin was observed for substantial tyrosinase inhibition and advocated for further investigation for drug development against tyrosinase-associated diseases.
- MeSH
- Agaricus enzymology MeSH
- Anthocyanins pharmacology MeSH
- Enzyme Inhibitors pharmacology MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Molecular Dynamics Simulation MeSH
- Molecular Docking Simulation MeSH
- Thermodynamics MeSH
- Monophenol Monooxygenase antagonists & inhibitors chemistry metabolism MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.
- MeSH
- Chlorophyll metabolism MeSH
- Carotenoids metabolism MeSH
- Protein Multimerization MeSH
- Mutation MeSH
- Energy Transfer MeSH
- Chloroplast Proteins chemistry genetics metabolism MeSH
- Arabidopsis Proteins chemistry genetics metabolism MeSH
- Protein Folding MeSH
- Synechocystis genetics metabolism MeSH
- Protein Binding MeSH
- Xanthophylls metabolism MeSH
- Zeaxanthins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
