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Background The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.

Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells.

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 &deg, C &times, 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

Nucleoside-diphosphate kinases (NME/Nm23/NDPK) are a family of evolutionary conserved enzymes present in all domains of life. The NDPKs transfer the terminal phosphate from a nucleoside triphosphate (mainly ATP) to all other NDPs. A huge interest for this enzyme started when it was discovered that a gene named nm23-H1 (non-metastatic clone 23, also known as NME1), responsible for suppression of metastasis in murine melanoma, is encoding one of the subunits of NDPK, NDPK A. The NME/Nm23/NDPK family members have been assigned a vast range of biological functions including metastasis suppression although the exact mechanism by which they execute their biological functions still remains to be unraveled. Numerous studies reported that proteins from evolutionary distinct organisms exhibit extraordinary similarity in their structures with their orthologs in mammals. Therefore, it is presumed that they have similar biochemical and biological functions which can, in consequence, serve as a new approach of studying human diseases. We performed a series of biochemical and biological tests which suggest that some of the NME genes/proteins in human are represented by a single ancestral type gene that already possesses many of the functions described in mammals. On the contrary, the NME genes that branched of very early in the evolutionary history seem to have changed significantly during metazoan evolution, and along did their protein function.

Sirtuin 3 (Sirt3) is the only member of sirtuin family linked to longevity in humans. In addition, important cellular and mitochondrial processes, including reactive oxygen species (ROS) generation are integrated through Sirt3. Furthermore, Sirt3 has a promising role in cancer tumorigenesis and treatment, but there have been controversies about its role as oncogene or tumor suppressor in different types of cancer. Breast cancer is the most frequent cancer among women, and ranks as the fifth cause of cancer death worldwide. Since breast cancer cells have strikingly reduced Sirt3 level, and even 20% of them have almost no detectable Sirt3 protein we wanted to examine the effect of de novo Sirt3 expression upon treatment with 95% O 2 (hyperoxia) in human MCF-7 breast cancer cells model. Since hypoxia is a hallmark of various tumors, we hypothesized that hyperoxia would have negative impact on cancer cells, thus providing therapeutic strategy against their tumorigenic properties. Therefore, in this study we have developed human MCF-7 breast cancer cells transfectants expressing the Sirt3 protein in order to test its potential in affecting the response of these cells upon the hyperoxic treatment, i.e. to decipher whether it sensitizes or makes these cancer cells more resistant to oxidative stress. De novo expression of Sirt3 decreased metabolic activity and cellular growth of MCF-7 cells, reduced expression of pro- angiogenic and epithelial mesenchymal transition genes, induced metabolic switch from glycolysis to oxidative phosphorylation, and decreased abundance of senescent cells. These effects were enhanced upon hyperoxic treatment: induction of DNA damage and upregulation of p53, with increase of ROS levels followed by mitochondrial and antioxidant dysfunction, resulted in additional reduction of metabolic activity and inhibition of cellular growth and survival. The mitigation of tumorigenic properties and enhancement of the susceptibility of the MCF-7 breast cancer cells to the hyperoxic treatment upon de novo Sirt3 expression, indicates that the impact of these factors, individually and in combination, should be further explored in vitro and particularly in vivo in breast cancer malignancies.

Yeast Saccharomyces cerevisiae cell wall is an extracellular organelle important for protection of the cell and its communication with the environment. In the cell wall structure, mannoproteins are covalently or noncovalently attached to a polysaccharide network that mostly consists of β-1, 3-glucan, β-1, 6-glucan and chitin. The cell wall is continually remodeled as yeast cells undergo life cycle or growth cycle transitions, which are therefore expected to be coupled with changes in expression of genes encoding cell wall proteins. However, little is known about differences in cell wall protein levels during different growth phases, such as logarithmic and stationary phase. We therefore performed relative quantification of yeast cell wall proteins in whole protein extracts of logarithmic and stationary cultures. Cell wall proteins were tagged at their genomic loci with a C-terminal hemagglutinin tag and were analyzed in whole protein extracts by western blot. Since the cell wall represents the non-soluble fraction of the cellular extract, validity of this method for extraction and quantification of cell wall proteins was established by constructing a set of diploid strains in which one or both copies of BGL2 gene, encoding a β-glucanase non-covalently bound to the cell wall, were tagged. Whole protein extracts of these strains showed a clear difference in signal intensity of Bgl2-HA band, thereby demonstrating that this method is appropriate for rapid and efficient extraction of cell wall proteins and is sensitive enough to detect a two-fold difference in protein level.

Multidrug resistance (MDR) is commonly known characteristic of many cancers and plays an important role in cancer treatment. Overexpression of ATP binding cassette (ABC) transporters called ABCB1 (MDR1/P-glycoprotein) and ABCG2 (BCRP) by cancer cells is responsible for this phenomenon, which makes this group of proteins a valuable target for cancer therapy by reversal of MDR. Salinomycin, naturally occurring potassium (K+) ionophore, was shown to inhibit P-gp transporter and to reverse MDR effectively. Salinomycin is a K+/H+ exchanger that can affect cation transport across different cellular membranes and thus impact on membranes’ bioenergetic performance, including mitochondrial membrane polarization and function. Based on the above-mentioned studies and anticancer activity of crown-ethers that act as K+ ionophores (previously published by our group), we showed that these compounds exhibit inhibitory effect towards ABC transporters, disrupt the potassium transport and modulate mitochondrial membrane transport as well. In this study, we showed the effect of adamantane-substituted monoaza- and diaza-18- crown-6 ether compounds on MDR reversal and their effect on mitochondrial membrane function, depending on their lipophilicity as well as on the chemical structure of the linker to adamantane moiety. Compounds that showed as the most potent P-gp and ABCG2 inhibitors were able to sensitize cancer cells to conventional chemotherapeutics paclitaxel and mitoxantrone, the substrates of P-gp and ABCG2 transporters, respectively. These data are promising and demonstrate a potential use of crown ethers in cancer therapy, by their ability to reverse MDR.

Notch signaling pathway is one of the main pathways directing different developmental processes, as its outcome also depends on cellular response and presence of interacting molecules. In hematopoietic cells, Notch has an important role in lymphocyte development. In this study, the aim was to analyze Notch signalization in different types of leukemia lymphocytes and the influence of poly(ADP-ribose) polymerase (PARP) activity on this process. PARP is an enzyme involved in DNA damage repair, posttranslational modification of transcription factors and chromatin structure changes. B and T leukaemia cells were treated with Notch (DAPT) and PARP (PJ-34) inhibitors, alone or in combination, for a prolonged period of time. All three types of cell lines showed high viability under the treatment and continued to proliferate. Analysis of gene and protein expression panel, involved in Notch and PARP pathways, showed ligand JAGGED1 expression increase after PARP1 inhibition in B cell lines and changes in Ikaros family members in both, B and T cell lines, regardless of the Notch signalling activity, after γ-secretase inhibition (DAPT). These data indicate that Notch and PARP inhibition, although not initiating differentiation in leukaemia cells analysed, induce changes in signalling circuits and chromatin modelling factors.

Delphinidin is a natural anthocyanidin that possesses numerous beneficial health effects. However, its effect in cisplatin (CP)- intoxicated mice is unknown. In this study we investigated the effect of delphinidin on CP- induced kidney injury. Male BALB/cN mice were treated orally by 1 and 5 mg/kg body weight of delphinidin daily for two days, 48 h after intraperitoneal injection of CP (13 mg/kg). CP treatment resulted in increased blood urea nitrogen and serum creatinine, with histopathological signs of renal tissue injury. CP administration also increased expression of oxidative/nitrosative stress markers 3- nitrotyrosine (3-NT) and heme oxygenase-1 (HO-1), as well as expression of proinflammatory cytokine tumor necrosis factor- alpha (TNF-α). Increased expression of caspase- 8, -9, and -3, with concomitant suppression od cyclin D1 expression and increased expression of p21, suggested inhibition of the cell cycle and induction of apoptosis in the kidneys. Surprisingly, treatment by delphinidin worsen CP-induced nephrotoxicity by increasing oxidative/nitrosative stress, inflammatory response, and apoptosis. Treatment by delphinidin suppressed CP-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and increased phosphorylation of c-Jun N- terminal kinase 1 (JNK1) and p38. Deterioration of CP-induced kidney injury by delphinidin was accompanied by suppression of efflux transporters multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2 (Mrp2) and increased platinum (Pt) levels in renal tissue. Our results suggest that delphinidin impairs renal Pt excretion, resulting in Pt accumulation and deterioration of CP-induced nephrotoxicity.

Honey is a natural sweetener with a complex composition. Honey features vary depending on the botanical source and geographical origin, as well as climatic, processing and storage conditions. Honey is a highly concentrated solution of a complex mixture of sugars ; namely fructose (38%) and glucose (31%) ; honey also contains other constituents such as minerals, proteins, vitamins, organic acid, flavonoids, phenolic acid, enzymes, and other phytochemicals in small quantities. Honey is a highly concentrated phenolic compounds are biologically active secondary metabolites from plants that act at molecular level and are potent natural antioxidants. Honey samples (n=97) were provided directly from beekeepers in 2017. The samples are from Croatia, Bosnia and Herzegovina, Serbia, Hungary and Italy. The physico-chemistry analysis of honey (HMF, electricar conductivity and moisture) was performing using the official methods of analysis (Codex Alimentarius Commission, 2001). The pH of the honey analysed was in the range 3, 38-6, 05, with the electrical conductivity was in the range 0, 13319-1, 738 mS/cm. Moisture in the range 14, 0-18, 8%. Moisture and pH are of great importance of honey texture, stability and shall life. HMF (Hydroxy Methyl Furfural) have been used as indicator of honey freshness in European because of their sensitivity to heat. An tested samples the honey had low levels HMF (O-6, 26 mg/kg honey). The quantification of total phenol in honey was preformed according to the Folin-Ciocalteu method as described by Singleton and Rossi (1965) using gallic acid as reference. An tested samples the honey was in the range 0, 043-0, 305 mg gallic acid per 100 g honey. The DPPH radical-scavenging assey based oh the abillity of antioxidant to block the 2, 2- difenil-1-picril-hidrazil radical.This relationship is confirm in this study ; the analysed honey present high antioxidant power, with a mean value of 67, 45% a range of 57, 2- 77, 7%. Honey samples were prepared and extracted with ethyl acetate according to the procedure by Ferreros et al., . The purity of the isolated flavonoids was tested by HPLC.

Dictyostelium amoebae can switch their polarity within twenty seconds, and thus undergo the fastest polarization reversal among eukaryotic cells. Signalling by small Rho GTPases plays an essential role in this process. In particular, Rac1 GTPases play a dual role in the regulation of actin assembly, which is a key determining factor of cell polarity. At the cell front, Rac1 activates the Scar/WAVE complex and thereby stimulates the Arp2/3-mediated actin polymerization. At the cell back, Rac1 regulates stability of the cell cortex by initiating formation of a complex containing IQGAP-related protein DGAP1 and a heterodimer of actin-bundling proteins cortexillins. I will discuss possible consequences of Rac1 interactions with multiple partners on its dynamics in living motile cells and present result obtained by imaging and modelling approaches. In this context, I will describe in more detail characterization of a fluorescent probe that selectively binds to the active form of Rac1, and demonstrate its excellent properties for live cell imaging. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase, and was selected on the basis of yeast two-hybrid screen, GST pull-down assay, and FRET measurements by fluorescence lifetime imaging microscopy (FLIM). An important feature of DPAKa(GBD)-DYFP probe is its finely graded intensity distribution along the entire plasma membrane, which enables measurements of the Rac1 activity in different parts of the membrane. Importantly, ectopic expression of DPAKa(GBD)-DYFP does not affect cell motility, cytokinesis and growth, thus enabling long-term imaging with negligible photobleaching and phototoxicity.

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks.

The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. ispartof: PLOS ONE vol:11 issue:1 ispartof: location:United States status: published

Stimulation of the receptor tyrosine kinase KIT by Stem Cell Factor (SCF) triggers activation of RAS and its downstream effectors. Proper KIT activation is essential for the maturation, survival and proliferation of mast cells. In addition, SCF activation of KIT is critical for recruiting mast cells to sites of infection or injury, where they release a mix of pro-inflammatory substances. RIN3, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), is highly expressed and enriched in human mast cells. SCF treatment of mast cells increased the amount of GTP-bound RAB5, and the degree of RAB5 activation correlated with the expression level of RIN3. At the same time, SCF caused the dissociation of a pre-formed complex of RIN3 with BIN2, a membrane bending protein implicated in endocytosis. Silencing of RIN3 increased the rate of SCF-induced KIT internalization, while persistent RIN3 over-expression led to KIT down regulation. These observations strongly support a role for RIN3 in coordinating the early steps of KIT endocytosis. Importantly, RIN3 also functioned as an inhibitor of mast cell migration toward SCF. Finally, we demonstrate that elevated RIN3 levels sensitize mastocytosis cells to treatment with a KIT tyrosine kinase inhibitor, suggesting the value of a two-pronged inhibitor approach for this difficult to treat malignancy. These findings directly connect KIT activation with a mast cell-specific RAS effector that regulates the cellular response to SCF and provide new insight for the development of more effective mastocytosis treatments.