Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.
- MeSH
- játra metabolismus MeSH
- kolorektální nádory * MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- makrolidy farmakologie MeSH
- nádory tračníku * MeSH
- subcelulární frakce metabolismus MeSH
- vakuolární protonové ATPasy * metabolismus MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- aktiny metabolismus MeSH
- buněčné jádro účinky léků metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- glykoly farmakologie MeSH
- lidé MeSH
- myosin typu I metabolismus MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- RNA-polymerasa II metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- vazba proteinů účinky léků MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Fibrates belong to a group of ligands of peroxisome proliferator-activated receptor alpha (PPARα), which play a role in the regulation of CYP epoxygenases and soluble epoxide hydrolase (sEH), key enzymes in the metabolism of biologically highly active epoxyeicosatrienoic acids (EETs). We demonstrated that low doses of fibrates stimulate proliferation of the MCF7 cell line, while high doses suppress it. The increase in cell proliferation was accompanied by an increase in CYP epoxygenases and decrease in sEH levels. The overall level of PPARα remained same after low-dose fibrate stimulation; however, there was a significant shift of the receptor to the cell nucleus. PPARα expression was further demonstrated by immunohistochemistry in both carcinoma and healthy breast tissue samples both in the cytoplasm and in the nuclei. We have also observed higher nuclear PPARα positivity in tumor tissues. Although our results obtained for MCF7 cells suggest the potential role of PPARα in cell proliferation, we did not find an association between nuclear localization of PPARα and the expression of proliferation marker Ki-67 in tumor tissues. The exact role of PPARα in carcinogenesis still remains unclear.
- MeSH
- antigen Ki-67 metabolismus MeSH
- deriváty kyseliny fibrové farmakologie MeSH
- epoxid hydrolasy metabolismus MeSH
- geny erbB-2 * MeSH
- inhibiční koncentrace 50 MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- nádory prsu metabolismus patologie MeSH
- PPAR alfa metabolismus MeSH
- proliferace buněk účinky léků MeSH
- pyrimidiny farmakologie MeSH
- receptory pro estrogeny metabolismus MeSH
- receptory progesteronu metabolismus MeSH
- rozpustnost MeSH
- subcelulární frakce metabolismus MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- transport proteinů účinky léků MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: In the present study, an attempt has been made for subtractive proteomic analysis approach for novel drug targets in Salmonella enterica subsp. enterica serover Typhi str.CT18 using computational tools. METHODS: Paralogous, redundant and less than 100 amino acid protein sequences were removed by using CD-HIT. Further detection of bacterial proteins which are non-homologous to host and are essential for the survival of pathogens by using BLASTp against host proteome and DEG`s, respectively. Comparative Metabolic pathways analysis was performed to find unique and common metabolic pathways. The non-redundant, non-homologous and essential proteins were BLAST against approved drug targets for drug targets while Psortb and CELLO were used to predict subcellular localization. RESULTS: There were 4473 protein sequences present in NCBI Database for Salmonella enterica subsp. enterica serover Typhi str. CT18 out of these 327 were essential proteins which were non-homologous to human. Among these essential proteins, 124 proteins were involved in 19 unique metabolic pathways. These proteins were further BLAST against approved drug targets in which 7 cytoplasmic proteins showed druggability and can be used as a therapeutic target. CONCLUSION: Drug targets identification is the prime step towards drug discovery. We identified 7 cytoplasmic druggable proteins which are essential for the pathogen survival and non-homologous to human proteome. Further in vitro and in vivo validation is needed for the evaluation of these targets to combat against salmonellosis.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny metabolismus MeSH
- mnohočetná bakteriální léková rezistence * MeSH
- počítačová simulace MeSH
- proteomika * MeSH
- Salmonella enterica účinky léků metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
A-kinase interacting protein 1 (AKIP1) has been shown to interact with a broad range of proteins involved in various cellular processes, including apoptosis, tumorigenesis, and oxidative stress suggesting it might have multiple cellular functions. In this study, we used an epitope-tagged AKIP1 and by combination of immunochemical approaches, microscopic methods and reporter assays we studied its properties. Here, we show that various levels of AKIP1 overexpression in HEK-293 cells affected not only its subcellular localization but also resulted in aggregation. While highly expressed AKIP1 accumulated in electron-dense aggregates both in the nucleus and cytosol, low expression of AKIP1 resulted in its localization within the nucleus as a free, non-aggregated protein. Even though AKIP1 was shown to interact with p65 subunit of NF-kappaB and activate this transcription factor, we did not observe any effect on NF-kappaB activation regardless of various AKIP1 expression level.
- MeSH
- adaptorové proteiny signální transdukční biosyntéza genetika MeSH
- buněčné jádro chemie metabolismus MeSH
- cytosol chemie metabolismus MeSH
- HEK293 buňky MeSH
- jaderné proteiny biosyntéza genetika MeSH
- lidé MeSH
- mitochondrie chemie metabolismus MeSH
- NF-kappa B analýza metabolismus MeSH
- regulace genové exprese MeSH
- subcelulární frakce chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.
- MeSH
- Arabidopsis účinky léků genetika mikrobiologie MeSH
- Botrytis účinky léků fyziologie MeSH
- fluorescence MeSH
- geneticky modifikované rostliny MeSH
- kořeny rostlin účinky léků genetika MeSH
- messenger RNA genetika metabolismus MeSH
- nemoci rostlin genetika mikrobiologie MeSH
- odolnost vůči nemocem účinky léků genetika MeSH
- peptidy farmakologie MeSH
- proteolýza účinky léků MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semenáček účinky léků růst a vývoj fyziologie MeSH
- Solanum lycopersicum mikrobiologie MeSH
- subcelulární frakce metabolismus MeSH
- transport RNA účinky léků genetika MeSH
- výhonky rostlin účinky léků genetika MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu2+. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.
- MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza MeSH
- komplexní sloučeniny metabolismus MeSH
- kumariny farmakologie MeSH
- lidé MeSH
- měď metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- subcelulární frakce metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.
- MeSH
- beta-katenin biosyntéza MeSH
- buněčná diferenciace účinky léků MeSH
- cytokiny biosyntéza MeSH
- epitelové buňky účinky léků MeSH
- interleukin-18 biosyntéza MeSH
- kaspasa 1 biosyntéza MeSH
- krysa rodu rattus MeSH
- Lacticaseibacillus rhamnosus * MeSH
- Lactobacillus casei * MeSH
- messenger RNA biosyntéza MeSH
- mikrofilamentové proteiny biosyntéza MeSH
- probiotika farmakologie MeSH
- regulace genové exprese účinky léků MeSH
- střevní sliznice cytologie účinky léků MeSH
- subcelulární frakce metabolismus MeSH
- toll-like receptory biosyntéza účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.
- MeSH
- fosforylace MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- metylace MeSH
- proteinarginin-N-methyltransferasy metabolismus fyziologie MeSH
- replikace viru fyziologie MeSH
- subcelulární frakce metabolismus MeSH
- ubikvitinace MeSH
- virus hepatitidy B enzymologie fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the process of male gamete development.
- MeSH
- buněčné jádro ultrastruktura MeSH
- dánio pruhované metabolismus MeSH
- spermatidy cytologie ultrastruktura MeSH
- spermatogeneze * MeSH
- subcelulární frakce metabolismus ultrastruktura MeSH
- testis cytologie ultrastruktura MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
