Search results for: phosphorylation

Authors: Dharshika Rajalingam, Jeffery W. Peng

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Acinetobacter baumannii is a challenging threat to global health, recognized as a multidrug-resistant pathogen. -lactamase is one of the principal resistant mechanisms developed by A. baumannii to survive against -lactam antibiotics. OXA24/40 is one of the types of -lactamases, a well-documented carbapenem hydrolyzing class D -lactamases (CHDL). It was revealed that OXA24/40 showed resistivity against doripenem, one of the carbapenems, by two different mechanisms as hydrolysis and -lactonization. Furthermore, it undergoes genetic mutations to broaden the -lactamase activity to survive against antibiotic environments. One of the crucial characterizations of prokaryotes to develop adaptation is post-translational modification (PTM), mainly phosphorylation. However, the PTM of OXA24/40 is an unknown feature, and the impact of PTM on antibiotic resistivity is yet to be explored. We approached these hypotheses using NMR and MS techniques and found that the OXA24/40 could be phosphorylated in vitro. The Ser81 at the active STFK motif of OXA24/40 of catalytic pocket was identified as the site of phosphorylation using 1D 31P NMR experiment, whereas S81 is required to form an acyl-enzyme complex between enzyme and -lactam antibiotics. The activity of completely phosphorylated OXA24/40 wild type against doripenem revealed that the phosphorylation of active Ser inactivates the -lactamases activity of OXA24/40. The 1D 1H CPMG NMR-based activity assay of phosphorylated OXA24/40 against doripenem confirmed that both deactivating mechanisms are inhibited by phosphorylation. Carbamylated Lysine at the active STFK motif is one of the critical features of CHDL required for the acylation and deacylation reactions of the enzyme. The 1D 13C NMR experiment confirmed that the K84 of phosphorylated OXA24/40 is de-carbamylated. Phosphorylation of OXA24/40 affects both active S81 and carbamylated K84 of OXA24 that are required for the resistivity of -lactamase. So, phosphorylation could be one of the reasons for the genetic mutation of OXA24/40 for the development of antibiotic resistivity. Further research can lead to an understanding of the effect of phosphorylation on the clinical mutants of the OXA24-like -lactamase family on the broadening of -lactamase activity.

Keywords: OXA24/40, phosphorylation, clinical mutants, resistivity

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Authors: Serap Pektas

Abstract:

Ataxia telangiectasia mutated (ATM) is a serine-threonine kinase, which is the major regulator of the DNA damage response. ATM is activated upon the formation of DNA double-strand breaks (DSBs) in the cells. ATM phosphorylates the proteins involved in apoptotic responses, cell cycle checkpoint control, DNA repair, etc. Tumor protein p53, known as p53 is one of these proteins that phosphorylated by ATM. Phosphorylation of p53 at Ser15 residue leads to p53 stabilization in the cells. Often enzymes activity is affected by hydrogen ion concentration (pH). In order to find the optimal pH range for ATM activity, steady-state kinetic assays were performed at acidic and basic pH ranges. Ser15 phosphorylation of p53 is determined by using ELISA. The results indicated that the phosphorylation rate was better at basic pH range compared with the acidic pH range. This could be due to enzyme stability, or enzyme-substrate interaction is pH dependent.

Keywords: ataxia telangiectasia mutated, DNA double strand breaks, DNA repair, tumor protein p53

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Authors: Malgorzata Krysiak, Anna Wegrzyn, Maciej Garstka, Radoslaw Mazur

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Under constantly fluctuating environmental conditions, the thylakoid membrane protein network evolved the ability to dynamically respond to changing biotic and abiotic factors. One of the most important protective mechanism is rearrangement of the chlorophyll-protein (CP) complexes, induced by protein phosphorylation. In a temperate climate, low temperature is one of the abiotic stresses that heavily affect plant growth and productivity. The aim of this study was to determine the role of LHCII antenna complex phosphorylation in the dark-chilling response. The study included an experimental model based on dark-chilling at 4 °C of detached chilling sensitive (CS) runner bean (Phaseolus coccineus L.) and chilling tolerant (CT) garden pea (Pisum sativum L.) leaves. This model is well described in the literature as used for the analysis of chilling impact without any additional effects caused by light. We examined changes in thylakoid membrane protein phosphorylation, interactions between phosphorylated LHCII (P-LHCII) and CP complexes, and their impact on the dynamics of photosystem II (PSII) under dark-chilling conditions. Our results showed that the dark-chilling treatment of CS bean leaves induced a substantial increase of phosphorylation of LHCII proteins, as well as changes in CP complexes composition and their interaction with P-LHCII. The PSII photochemical efficiency measurements showed that in bean, PSII is overloaded with light energy, which is not compensated by CP complexes rearrangements. On the contrary, no significant changes in PSII photochemical efficiency, phosphorylation pattern and CP complexes interactions were observed in CT pea. In conclusion, our results indicate that different responses of the LHCII phosphorylation to chilling stress take place in CT and CS plants, and that kinetics of LHCII phosphorylation and interactions of P-LHCII with photosynthetic complexes may be crucial to chilling stress response. Acknowledgments: presented work was financed by the National Science Centre, Poland grant No.: 2016/23/D/NZ3/01276

Keywords: LHCII, phosphorylation, chilling stress, pea, runner bean

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Authors: Tarana Siddika, Ilka U. Heinemann, Patrick O’Donoghue

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Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Threonine308, Serine473), yet cell stimulation also activates many other kinases and fails to differentiate the effect of the two main activating sites of AKT1 on downstream substrate phosphorylation and cell growth. While both AKT1 activating sites are associated with disease and used as clinical markers, in some cancers, high levels of Threonine308 phosphorylation are associated with poor prognosis while in others poor survival correlates with high Serine473 levels. To produce cells with specific AKT1 activity, a system was developed to deliver active AKT1 to human cells. AKT1 phospho-variants were produced from Escherichia coli with programmed phosphorylation by genetic code expansion. Tagging of AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) helped to enter AKT1 proteins in mammalian cells. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308, TAT-pAKT1S473 and TAT-pAKT1T308S473 proteins induced selective phosphorylation of the known AKT1 substrate GSK-3αβ, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Serine240/244 in transfected cells. Increase in cell growth and proliferation was observed due to the transfection of different phosphorylated AKT1 protein variants compared to cells with TAT-AKT1 protein. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on specific AKT1 activity and phosphorylation.

Keywords: cell penetrating peptide, cell signaling, protein kinase b (AKT1), phosphorylation

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Authors: Wenxing Chen, Guangying Chen, Yin Lu, Shile Huang

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Cryptotanshinone (CPT), a fat-soluble tanshinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit mTOR pathway, resulting in inhibition of cancer cell proliferation. However, the molecular mechanism how CPT acts on mTOR is unknown. Here, cancer cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while phosphorylation of AMPK and TSC2 was activated, suggesting that CPT inhibition of mTOR maybe due to activating upstream of mTOR, AMPK, but not directly binding to and inhibiting mTOR. Further results indicated that Compound C, inhibitor of AMPK, could partially reversed CPT inhibition effect on cancer cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4EBP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with MEF cells with AMPK positive, MEF cells with AMPK knock out are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively much. Furthermore, downexpression of TSC2 slightly recovered expression of 4EBP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not affect expression of TSC1 by CPT. Collectively, the above-mentioned results suggest that CPT inhibited mTOR pathway mostly was due to activation of AMPK-TSC2 pathway rather than specific inhibition of mTOR and then induction of subsequent lethal cellular effect.

Keywords: cryptotanshinone, AMPK, TSC2, mTOR, cancer cells

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Authors: Hakam Alkhateeb

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Oleuropein, the main constituent of leaves and fruits of the olive tree, has been demonstrated to exert beneficial effects on parameters relevant to the normal homeostatic mechanisms of glucose regulation in rat skeletal muscle. However, the antidiabetic effect of oleuropein, to our knowledge, has not been examined. Therefore, in this study, we examined whether oleuropein ameliorated palmitate-induced insulin resistance in skeletal muscle. To examine this question, insulin resistance was rapidly induced by incubating (12h) soleus muscle with a high concentration of palmitate(2mM). Subsequently, we attempted to restore insulin sensitivity by incubating (12h) muscles with oleuropien (1.5mM), while maintaining high concentrations of palmitate. Palmitate treatment for 12 h reduced insulin-stimulated glucose transport, GLUT4 translocationandAS160 phosphorylation. Oleuropein treatment (12 h) fully restoredinsulin-stimulated glucose transport, GLUT4translocationandAS160 phosphorylation. Inhibition of PI3K phosphorylation with wortmannin (1µM)did not affect the oleuropein-induced improvements in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. These results suggested that the improvements in these parameters cannot account for activating PI3K pathway. Taken altogether, it appears that oleuropein, through activation of another pathway like activated protein kinase (AMPK), may provide a possible strategy by which they ameliorate palmitate-induced insulin resistance in skeletal muscles.

Keywords: AS160, diabetes, GLUT4, oleuropein

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Authors: Hainan Chen, Jina Qing, Xiao Zhu, Ling Gao, Ampadu O. Jackson, Min Zhang, Kai Yin

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Objective: Editing macrophage activation to dampen inflammatory diseases by promoting the repolarization of inflammatory (M1) macrophages to anti-inflammatory (M2) macrophages is highly associated with mitochondrial respiration. Recent studies have suggested that mitochondrial apolipoprotein A-1 binding protein (APOA1BP) was essential for the cellular metabolite NADHX repair to NADH, which is necessary for the mitochondrial function. The exact role of APOA1BP in the repolarization of M1 to M2, however, is uncertain. Material and method: THP-1-derived macrophages were incubated with LPS (10 ng/ml) or/and IL-4 (100 U/ml) for 24 hours. Biochemical parameters of oxidative phosphorylation and M1/M2 markers were analyzed after overexpression of APOA1BP in cells. Results: Compared with control and IL-4-exposed M2 cells, APOA1BP was downregulated in M1 macrophages. APOA1BP restored the decline in mitochondrial function to improve metabolic and phenotypic reprogramming of M1 to M2 macrophages. Blocking oxidative phosphorylation by oligomycin blunts the effects of APOA1BP on M1 to M2 repolarization. Mechanistically, LPS triggered the hydration of NADH and increased its hydrate NADHX which inhibit cellular NADH dehydrogenases, a key component of electron transport chain for oxidative phosphorylation. APOA1BP decreased the level of NADHX via converting R-NADHX to biologically useful S-NADHX. The mutant of APOA1BP aspartate188, the binding site of NADHX, fail to repair oxidative phosphorylation, thereby preventing repolarization. Conclusions: Restoring mitochondrial function by increasing mitochondrial APOA1BP might be useful to improve the reprogramming of inflammatory macrophages into anti-inflammatory cells to control inflammatory diseases.

Keywords: inflammatory diseases, macrophage repolarization, mitochondrial respiration, apolipoprotein A-1 binding protein, NADHX, NADH

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Authors: Hae-Jun Lee, Ji-Sun Shin, Kyung-Tae Lee

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Potentilla species (Rosasease) have been used in traditional medicine to treat different ailment, disease or malady. In this study, we investigated the anti-inflammatory effects of ethanol extracts of NUTT (EPP) in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages and septic mice. EPP suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced Raw 264.7 macrophages. Consistent with these observations, EPP reduced the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by downregulation of their promoter activities. EPP inhibited tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) at production and mRNA levels. Molecularly, EPP attenuated the LPS-induced transcriptional activity, and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α (IκB-α) degradation and IκB kinase-α/β (IKK-α/β) phosphorylation. Furthermore, EPP suppressed the LPS-induced activation of activator protein-1 (AP-1) by reducing the expression of c-Fos and c-Jun in nuclear. EPP also reduced the phosphorylation of mitogen-activated protein kinase (MAPK), such as p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). In a sepsis model, pretreatment with EPP reduced the LPS-induced lethality. Collectively, these results suggest that the anti-inflammatory effects of EPP were associated with the suppression of NF-κB and AP-1 activation, and support its possible therapeutic role for the treatment of sepsis.

Keywords: anti-inflammation, activator protein-1, nuclear factor κB, Potentilla paradoxa Nutt

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Authors: Chowon Kim, Yoo-Kyung Kim, Kyeong Yeon Park, Hyun-Shik Lee

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Perfluoroalkyl compounds (PFCs) are globally distributed synthetic compounds that are known to adversely affect human health. Developmental toxicity assessment of PFCs is important to facilitate the evaluation of their environmental impact. In the present study, we assessed the developmental toxicity and teratogenicity of PFCs with different numbers of carbon atoms on Xenopus embryogenesis. An initial frog embryo teratogenicity assay-Xenopus (FETAX) assay was performed that identified perfluorohexanoic (PFHxA) and perfluoroheptanoic (PFHpA) acids as potential teratogens and developmental toxicants. The mechanism underlying this teratogenicity was also investigated by measuring the expression of tissue-specific biomarkers such as phosphotyrosine‑binding protein, xPTB (liver); NKX2.5 (heart); and Cyl18 (intestine). Whole‑mount in situ hybridization, reverse transcriptase‑polymerase chain reaction (RT-PCR), and histologic analyses detected severe defects in the liver and heart following exposure to PFHxA or PFHpA. In addition, immunoblotting revealed that PFHpA significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), while PFHxA slightly increased these, as compared with the control. These results suggest that PFHxA and PFHpA are developmental toxicants and teratogens, with PFHpA producing more severe effects on liver and heart development through the induction of ERK and JNK phosphorylation.

Keywords: PFCs, ERK, JNK, xenopus

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Authors: Li Chen, Feng Zhang, Shizhong Zheng

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SaikosaponinD (SSD) is a major component of saikosaponins isolated from Bupleurumfalactum. Our current study was to examine the toxic effect of SSD on liver cells and explore the possible mechanism. The results demonstrated that SSD induced mouse liver injury and led to apoptosis in LO2 cells. HE staining and TUNEL analyses showed that SSD stimulated liver injury and hepatocyte apoptosis in vivo. Subsequent experiments showed that SSD down-regulated Bcl-2 but up-regulated Bax. In vitro, SSD-treated LO2 cells exhibited apparent down-regulated expression of p-p38. Moreover, PDGF-βR agonist PDGF-BB alone significantly upregulated p38 phosphorylation, while combined with SSD, p38 phosphorylation expression was reduced. Furthermore, shRNA-mediated PDGF-βR knockdown augmented the inactivation of p-p38 and Bcl2 but abrogated the activation of Bax, these results were more obvious when shRNA combined with SSD. These data indicated that SSD stimulated liver injury and apoptosis in hepatocytes and PDGF-βR /p38 pathway may be the potential mechanistic.

Keywords: saikosaponin D, hepatotoxicity, liver injury, apoptosis, platelet-derived growth factor-β receptor, p38

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Authors: N. S. Vassilieva-Vashakmadze, R. A. Gakhokidze, I. M. Khachatryan

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In the first part of the paper it is shown that both the depolarization of the cytoplasmic membrane of rods observed in invertebrates and hyperpolarization characteristic of vertebrates on the light may activate the functioning of ion (Na+) channels of cytoplasmic membrane of rods and thus provide the emergence of nerve impulse and its transfer to the neighboring neuron etc. In the second part, using the quantum mechanical program for modeling of the molecular processes, we got a clear picture demonstrating the effect of charged phosphate groups on the protein components of α-helical subunits of the visual rhodopsin receptor. The analysis shows that the phosphorylation of terminal amino acid of seventh α-helical subunits of the visual rhodopsin causes a redistribution of electron density on the atoms, i.e. polarization of subunits, also the changing the configuration of the nuclear subsystem, which corresponds to the deformation process in the molecule. Based on the use of models it can be concluded that this system has an internal relationship between polarization and deformation processes that indicates on the allosteric effect. The allosteric effect is based on quantum-mechanical principle of the self-consistency of the molecules.

Keywords: membrane potential, ion channels, visual rhodopsin, allosteric effect

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Authors: Manuel A. Alonso-Tarajano, Roberto Mosca, Patrick Aloy

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Protein kinases participate in a myriad of cellular processes of major biomedical interest. The in vivo substrate specificity of these enzymes is a process determined by several factors, and despite several years of research on the topic, is still far from being totally understood. In the present work, we have quantified the contributions to the kinase substrate specificity of i) the phosphorylation sites and their surrounding residues in the sequence and of ii) the association of kinases to adaptor or scaffold proteins. We have used position-specific scoring matrices (PSSMs), to represent the stretches of sequences phosphorylated by 93 families of kinases. We have found negative correlations between the number of sequences from which a PSSM is generated and the statistical significance and the performance of that PSSM. Using a subset of 22 statistically significant PSSMs, we have identified specificity determinant residues (SDRs) for 86% of the corresponding kinase families. Our results suggest that different SDRs can function as positive or negative elements of substrate recognition by the different families of kinases. Additionally, we have found that human proteins with known function as adaptors or scaffolds (kAS) tend to interact with a significantly large fraction of the substrates of the kinases to which they associate. Based on this characteristic we have identified a set of 279 potential adaptors/scaffolds (pAS) for human kinases, which is enriched in Pfam domains and functional terms tightly related to the proposed function. Moreover, our results show that for 74.6% of the kinase– pAS association found, the pAS colocalize with the substrates of the kinases they are associated to. Finally, we have found evidence suggesting that the association of kinases to adaptors and scaffolds, may contribute significantly to diminish the in vivo substrate crossed- specificity of protein kinases. In general, our results indicate the relevance of several SDRs for both the positive and negative selection of phosphorylation sites by kinase families and also suggest that the association of kinases to pAS proteins may be an important factor for the localization of the enzymes with their set of substrates.

Keywords: kinase, phosphorylation, substrate specificity, adaptors, scaffolds, cellular colocalization

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Authors: Rajapaksha Gedara Prasad Tharanga Jayasooriya, Matharage Gayani Dilshara, Yung Hyun Choi, Gi-Young Kim

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Camptothecin (CPT) is a quinolone alkaloid which inhibits DNA topoisomerase I that induces cytotoxicity in a variety of cancer cell lines. We previously showed that CPT effectively inhibited invasion of prostate cancer cells and also combined treatment with subtoxic doses of CPT and TNF-related apoptosis-inducing ligand (TRAIL) potentially enhanced apoptosis in a caspase-dependent manner in hepatoma cancer cells. Here, we found that treatment with CPT caused an irreversible cell cycle arrest in the G2/M phase. CPT-induced cell cycle arrest was associated with a decrease in protein levels of cell division cycle 25C (Cdc25C) and increased the level of cyclin B and p21. The CPT-induced decrease in Cdc25C was blocked in the presence of proteasome inhibitor MG132, thus reversed the cell cycle arrest. In addition to that treatment of CPT-increased phosphorylation of Cdc25C was the resulted of activation of checkpoint kinase 2 (Chk2), which was associated with phosphorylation of ataxia telangiectasia-mutated. Interestingly CPT induced G2/M phase of the cell cycle arrest is reactive oxygen species (ROS) dependent where ROS inhibitors NAC and GSH reversed the CPT-induced cell cycle arrest. These results further confirm by using transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2) since it regulates the production of ROS. Our data reveal that treatment of siNrf2 increased the ROS level as well as further increased the CPT induce G2/M phase cell cycle arrest. Our data also indicate CPT-enhanced cell cycle arrest through the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) pathway. Inhibitors of ERK and JNK more decreased the Cdc25C expression and protein expression of p21 and cyclin B. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in G2/M arrest by CPT.

Keywords: camptothecin, cell cycle, checkpoint kinase 2, nuclear factor-erythroid 2-related factor 2, reactive oxygen species

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Authors: Nootchanat Mairuae, Walaiporn Tongjaroenbuangam, Chalisa Louicharoen Cheepsunthorn, Poonlarp Cheepsunthorn

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The aim of this study was to investigate the anti-oxidative effect, the anti-inflammatory effects, and the molecular mechanisms of okra (Abelmoschus esculentus Linn.) on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The BV2 cells were treated with LPS in the presence or absence of okra. Reactive oxygen species (ROS) and nitric oxide (NO) production were measured using the ROS detection reagent DCF-DA and the Griess reaction, respectively. The phosphorylation levels of nuclear factor-kappa B (NF-kB) p65 was detected by Western blot assay. Treatment of BV2 microglia cells with okra was found to significantly suppress the LPS-induced inflammatory mediator NO as well as ROS compared to untreated cells. The levels of LPS-induced NF-kB p65 phosphorylation were significantly decreased following okra treatment too. These results show that okra exerts anti-oxidative and anti-inflammatory effects in LPS-stimulated BV2 microglial cells by suppressing the NF-κB pathway. This suggests okra might be a valuable agent for treatment of anti-neuroinflammatory diseases mediated by microglial cells.

Keywords: Abelmoschus esculentus Linn, microglia, neuroinflammation, reactive oxygen spicy

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Authors: Eun Young Choi, So Hui Choe, Jin Yi Hyeon, Ji Young Jin, Bo Ram Keum, Jong Min Lim, Hyung Rae Cho, Kwang Keun Cho, In Soon Choi

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This research analyzed the effect of β-glucan that is expected to alleviate the production of inflammatory mediator in macrophagocyte, which was processed by the lipopolysaccharide (LPS) of Escherichia, a pathogen related to allergy. The incubated layer was used for nitric oxide (NO) analysis. The DNA-binding activation of the small unit of NF-κB was measured using ELISA-based kit. In RAW264.7 cells that were vitalized by E.coli LPS, β-glucan inhibited both the combatant and rendering phases of inducible NO synthase (iNOS)-derived NO. β-glucan increased the expression of heme oxygenase-1 (HO-1) in the cell that was stimulated by E.coli LPS, and HO-1 activation was inhibited by SnPP. This shows that NO production induced by LPS is related to the inhibition effect of β-glucan. The phosphorylation of JNK and p38 induced by LPS were not influenced by β-glucan, and IκB-α decomposition was not influenced either. Instead, β-glucan remarkably inhibited the phosphorylation of STAT1 that was induced by E.coli LPS. Overall, β-glucan inhibited the production of NO in macrophagocyte that was vitalized by E.coli LPS through HO-1 induction and STAT1 pathways inhibition in this research. As the host inflammation reaction control by β-glucan weakens the progress of allergy, β-glucan can be used as an effective treatment method.

Keywords: β-glucan, lipopolysaccharide (LPS), nitric oxide (NO), RAW264.7 cells, STAT1

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Authors: Pramod Somvanshi, Manu Tomar, K. V. Venkatesh

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Insulin and glucagon are responsible for homeostasis of key plasma metabolites like glucose, amino acids and fatty acids in the blood plasma. These hormones act antagonistically to each other during the secretion and signaling stages. In the present work, we analyze the effect of macronutrients on the response from integrated insulin and glucagon signaling pathways. The insulin and glucagon pathways are connected by DAG (a calcium signaling component which is part of the glucagon signaling module) which activates PKC and inhibits IRS (insulin signaling component) constituting a crosstalk. AKT (insulin signaling component) inhibits cAMP (glucagon signaling component) through PDE3 forming the other crosstalk between the two signaling pathways. Physiological level of anabolism and catabolism is captured through a metric quantified by the activity levels of AKT and PKA in their phosphorylated states, which represent the insulin and glucagon signaling endpoints, respectively. Under resting and starving conditions, the phosphorylation metric represents homeostasis indicating a balance between the anabolic and catabolic activities in the tissues. The steady state analysis of the integrated network demonstrates the presence of a bistable response in the phosphorylation metric with respect to input plasma glucose levels. This indicates that two steady state conditions (one in the homeostatic zone and other in the anabolic zone) are possible for a given glucose concentration depending on the ON or OFF path. When glucose levels rise above normal, during post-meal conditions, the bistability is observed in the anabolic space denoting the dominance of the glycogenesis in liver. For glucose concentrations lower than the physiological levels, while exercising, metabolic response lies in the catabolic space denoting the prevalence of glycogenolysis in liver. The non-linear positive feedback of AKT on IRS in insulin signaling module of the network is the main cause of the bistable response. The span of bistability in the phosphorylation metric increases as plasma fatty acid and amino acid levels rise and eventually the response turns monostable and catabolic representing diabetic conditions. In the case of high fat or protein diet, fatty acids and amino acids have an inhibitory effect on the insulin signaling pathway by increasing the serine phosphorylation of IRS protein via the activation of PKC and S6K, respectively. Similar analysis was also performed with respect to input amino acid and fatty acid levels. This emergent property of bistability in the integrated network helps us understand why it becomes extremely difficult to treat obesity and diabetes when blood glucose level rises beyond a certain value.

Keywords: bistability, diabetes, feedback and crosstalk, obesity

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Authors: Reem Al-Shidhani, Isabelle S. R. Storer, Michael J. Bromley, Lydia Tabernero

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Invasive fungal diseases are an increasing global health concern that contributes to the high mortality rates in immunocompromised patients. The rising of antifungal resistance severely lowers the efficacy of the limited antifungal agents available. New antifungal drugs that target new mechanisms are necessary to tackle the current shortfalls. Amongst post- modifications, phosphorylation is a predominant and an outstanding protein alteration in all eukaryotes. In fungi, protein phosphorylation plays a vital role in many signal transduction pathways, including cell cycle, cell growth, metabolism, transcription, differentiation, proliferation, and virulence. The investigation of Aspergillus fumigatus phosphatases revealed seven genes essential for viability. Inhibiting one of these phosphatases is a new interesting route to develop novel antifungal drugs. In this study, we carried out an early drug discovery process targeting oneessential phosphatase, GlcA. Here, we report the identification of new GlcA inhibitors that show antifungal activity. These important finding open a new avenue to the development of novel antifungals to expand the current narrow arsenal of clinical candidates.

Keywords: invasive fungal diseases, phosphatases, GlcA, competitive inhibitors

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Authors: Qiang Xu, Xingxin Wu, Wenjie Guo, Xingqi Wang, Yang Sun, Renxiang Tan

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The phosphatase SHP-2 is known to exert regulatory activities on cytokine receptor signaling and the dysregulation of SHP-2 has been implicated in the pathogenesis of a variety of diseases. Here we report several small molecule regulators of SHP-2 for the treatment of T cell-mediated diseases. The new cyclodepsipeptide trichomides A, isolated from the fermentation products of Trichothecium roseum, increased the phosphorylation of SHP-2 in activated T cells, and ameliorated contact dermatitis in mice. The trichomides A’s effects were significantly reversed by using the SHP-2-specific inhibitor PHPS1 or T cell-conditional SHP-2 knockout mice. Another compound is a cerebroside Fusaruside isolated from the endophytic fungus Fusarium sp. IFB-121. Fusaruside also triggered the tyrosine phosphorylation of SHP-2, which provided a possible mean of selectively targeting STAT1 for the treatment of Th1 cell-mediated inflammation and led to the discovery of the non-phosphatase-like function of SHP-2. Namely, the Fusaruside-activated pY-SHP-2 selectively sequestrated the cytosolic STAT1 to prevent its recruitment to IFN-R, which contributed to the improvement of experimental colitis in mice. Blocking the pY-SHP-2-STAT1 interaction, with SHP-2 inhibitor NSC-87877 or using T cells from conditional SHP-2 knockout mice, reversed the effects of fusaruside. Furthermore, the fusaruside’s effect is independent of the phosphatase activity of SHP-2, demonstrating a novel role for SHP-2 in regulating STAT1 signaling and Th1-type immune responses.

Keywords: SHP-2, small molecules, T cell, T cell-mediated diseases

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Authors: Maricarmen Herrera, Pierre Chymkowitch, Joe Robertson, Jens Eriksson, Jorrit Enserink

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tRNA genes are transcribed by RNA polymerase III. During recent years, it has become clear that tDNA transcription fluctuates during the cell cycle. However, the mechanism by which the cell cycle controls the amplitude of tDNA transcription remains unknown. We found that the cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to sharply increase tRNA synthesis during a brief interval in the cell cycle. We show that Cdk1 promotes the interaction of TFIIIB with TFIIIC, that it stimulates the recruitment of TFIIIC to tRNA genes, that it prevents the formation of an overly stable TFIIIB-tDNA complex and that it augments the dynamics of RNA polymerase III. Furthermore, we identify Bdp1 as a novel Cdk1 substrate, and phosphorylation of Bdp1 is required for the cell cycle-dependent increase in tDNA transcription. In addition, we show that phosphorylation of the Cdk1 substrate Nup60 mediates formation of a Nup60-Nup2 complex at tRNA genes, which is also required for cell cycle-dependent tDNA transcription. Together, our findings indicate that Cdk1 activity gates tRNA synthesis by regulating the dynamics of the TFIIIB-TFIIIC-RNAPIII complex, and that it may promote the formation of a nuclear pore microenvironment conducive to efficient tDNA transcription.

Keywords: Cdk1, cell cycle, RNAPIII machinery, tRNA

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Authors: Seyedahmad Hosseini, Mohammadjavad Sotoudeheian

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Introduction: Allergic asthma is a heterogeneous immuno-inflammatory disease based on Th-2-mediated inflammation. Histopathologic abnormalities of the airways characteristic of asthma include epithelial damage and subepithelial collagen deposition. Objectives: Human bronchial epithelial cell genome expression of TNF‑α, IL‑6, ICAM‑1, VCAM‑1, nuclear factor (NF)‑κB signaling pathways up-regulate during inflammatory cascades. Moreover, immunofluorescence assays confirmed the nuclear translocation of NF‑κB p65 during inflammatory responses. An absolute LDH leakage assays suggestedLPS-inducedcells injury, and the associated mechanisms are co-incident events. LPS-induced phosphorylation of ERKand JNK causes inflammation in epithelial cells through inhibition of ERK and JNK activation and NF-κB signaling pathway. Furthermore, the inhibition of NF-κB mRNA expression and the nuclear translocation of NF-κB lead to anti-inflammatory events. Likewise, activation of SUMF2 which inhibits IL-13 and reduces Th2-cytokines, NF-κB, and IgE levels to ameliorate asthma. On the other hand, TNFα-induced mucus production reduced NF-κB activation through inhibition of the activation status of Rac1 and IκBα phosphorylation. In addition, bradykinin B2 receptor (B2R), which mediates airway remodeling, regulates through NF-κB. Bronchial B2R expression is constitutively elevated in allergic asthma. In addition, certain NF-κB -dependent chemokines function to recruit eosinophils in the airway. Besides, bromodomain containing 4 (BRD4) plays a significant role in mediating innate immune response in human small airway epithelial cells as well as transglutaminase 2 (TG2), which is detectable in saliva. So, the guanine nucleotide-binding regulatory protein α-subunit, Gα16, expresses a κB-driven luciferase reporter. This response was accompanied by phosphorylation of IκBα. Furthermore, expression of Gα16 in saliva markedly enhanced TNF-α-induced κB reporter activity. Methods: The applied method to form NF-κB activation is the electromobility shift assay (EMSA). Also, B2R-BRD4-TG2 complex detection by immunoassay method within saliva with EMSA of NF-κB activation may be a novel biomarker for asthma diagnosis and follow up. Conclusion: This concept introduces NF-κB signaling pathway as potential asthma biomarkers and promising targets for the development of new therapeutic strategies against asthma.

Keywords: NF-κB, asthma, saliva, T-helper

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Authors: Sandeep Kaur, Jenny Vuong, Marcel Julliard, Sean O'Donoghue

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Time-series data are useful for modelling as they can enable model-evaluation. However, when reconstructing models from phosphoproteomic data, often non-exact methods are utilised, as the knowledge regarding the network structure, such as, which kinases and phosphatases lead to the observed phosphorylation state, is incomplete. Thus, such reactions are often hypothesised, which gives rise to uncertainty. Here, we propose a framework, implemented via a web-based tool (as an extension to Minardo), which given time-series phosphoproteomic datasets, can generate κ models. The incompleteness and uncertainty in the generated model and reactions are clearly presented to the user via the visual method. Furthermore, we demonstrate, via a toy EGF signalling model, the use of algorithmic verification to verify κ models. Manually formulated requirements were evaluated with regards to the model, leading to the highlighting of the nodes causing unsatisfiability (i.e. error causing nodes). We aim to integrate such methods into our web-based tool and demonstrate how the identified erroneous nodes can be presented to the user via the visual method. Thus, in this research we present a framework, to enable a user to explore phosphorylation proteomic time-series data in the context of models. The observer can visualise which reactions in the model are highly uncertain, and which nodes cause incorrect simulation outputs. A tool such as this enables an end-user to determine the empirical analysis to perform, to reduce uncertainty in the presented model - thus enabling a better understanding of the underlying system.

Keywords: κ-models, model verification, time-series phosphoproteomic datasets, uncertainty and error visualisation

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Authors: Ireneusz Majsterek, Dariusz Pytel, J. Alan Diehl

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PKR-like ER kinase (PERK) is serine/threonie endoplasmic reticulum (ER) transmembrane kinase activated during ER-stress. PERK can activate signaling pathways known as unfolded protein response (UPR). Attenuation of translation is mediated by PERK via phosphorylation of eukaryotic initiation factor 2α (eIF2α), which is necessary for translation initiation. PERK activation also directly contributes to activation of Nrf2 which regulates expression of anti-oxidant enzymes. An increased phosphorylation of eIF2α has been reported in Alzheimer disease (AD) patient hippocampus, indicating that PERK is activated in this disease. Recent data have revealed activation of PERK signaling in non-Hodgkins lymphomas. Results also revealed that loss of PERK limits mammary tumor cell growth in vitro and in vivo. Consistent with these observations, activation of UPR in vitro increases levels of the amyloid precursor protein (APP), the peptide from which beta-amyloid plaques (AB) fragments are derived. Finally, proteolytic processing of APP, including the cleavages that produce AB, largely occurs in the ER, and localization coincident with PERK activity. Thus, we expect that PERK-dependent signaling is critical for progression of many types of diseases (human cancer, neurodegenerative disease and other). Therefore, modulation of PERK activity may be a useful therapeutic target in the treatment of different diseases that fail to respond to traditional chemotherapeutic strategies, including Alzheimer’s disease. Our goal will be to developed therapeutic modalities targeting PERK activity.

Keywords: PERK kinase, small molecule inhibitor, neurodegenerative disease, Alzheimer’s disease

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Authors: Jin Boo Jeong

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In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells. DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3β. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of β-catenin and TCF4, and β–catenin/TCF-dependent luciferase activity. Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3β, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A3B03931713).

Keywords: anticancer, calyx of persimmon, cyclin D1, Diospyros kaki Thunb., human colorectal cancer

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Authors: Pallab Datta, Jyotirmoy Chatterjee, Santanu Dhara

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Over the past few years, phosphorous containing polymers have received widespread attention for applications such as high performance optical fibers, flame retardant materials, drug delivery and tissue engineering. Being pentavalent, phosphorous can exist in different chemical environments in these polymers which increase their versatility. In human biochemistry, phosphorous based compounds exert their functions both in soluble and insoluble form occurring as inorganic or as organophosphorous compounds. Specifically in case of biomacromolecules, phosphates are critical for functions of DNA, ATP, phosphoproteins, phospholipids, phosphoglycans and several coenzymes. Inspired by the role of phosphorous in functional biomacromolecules, design and synthesis of biomimetic materials are thus carried out by several authors to study macromolecular function or as substitutes in clinical tissue regeneration conditions. In addition, many regulatory signals of the body are controlled by phoshphorylation of key proteins present either in form of growth factors or matrix-bound scaffold proteins. This inspires works on synthesis of phospho-peptidomimetic amino acids for understanding key signaling pathways and this is extended to obtain molecules with potentially useful biological properties. Apart from above applications, phosphate groups bound to polymer backbones have also been demonstrated to improve function of osteoblast cells and augment performance of bone grafts. Despite the advantages of phosphate grafting, however, there is limited understanding on effect of degree of phosphorylation on macromolecular physicochemical and/or biological properties. Such investigations are necessary to effectively translate knowledge of macromolecular biochemistry into relevant clinical products since they directly influence processability of these polymers into suitable scaffold structures and control subsequent biological response. Amongst various techniques for fabrication of biomimetic scaffolds, nanofibrous scaffolds fabricated by electrospinning technique offer some special advantages in resembling the attributes of natural extracellular matrix. Understanding changes in physico-chemical properties of polymers as function of phosphorylation is therefore going to be crucial in development of nanofiber scaffolds based on phosphorylated polymers. The aim of the present work is to investigate the effect of phosphorous grafting on the electrospinning behavior of polymers with aim to obtain biomaterials for bone regeneration applications. For this purpose, phosphorylated derivatives of two polymers of widely different electrospinning behaviors were selected as starting materials. Poly(vinyl alcohol) is a conveniently electrospinnable polymer at different conditions and concentrations. On the other hand, electrospinning of chitosan backbone based polymers have been viewed as a critical challenge. The phosphorylated derivatives of these polymers were synthesized, characterized and electrospinning behavior of various solutions containing these derivatives was compared with electrospinning of pure poly (vinyl alcohol). In PVA, phosphorylation adversely impacted electrospinnability while in NMPC, higher phosphate content widened concentration range for nanofiber formation. Culture of MG-63 cells on electrospun nanofibers, revealed that degree of phosphate modification of a polymer significantly improves cell adhesion or osteoblast function of cultured cells. It is concluded that improvement of cell response parameters of nanofiber scaffolds can be attained as a function of controlled degree of phosphate grafting in polymeric biomaterials with implications for bone tissue engineering applications.

Keywords: bone regeneration, chitosan, electrospinning, phosphorylation

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Authors: A. G. Miah, U. Salma, K. Schellander

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Relaxin was first described in 1926 by Frederick Hisaw. Previously it was considered as only the hormone of pregnant mammals due to its important roles in pregnancy and parturition. From the last decade, the physiological role of relaxin in male reproduction has been given experimental attention, and the results have made it clear that relaxin can no longer be considered strictly as only the hormone of female reproduction. The accessory glands (specially, the prostate glands) of the male reproductive system are the source of seminal relaxin, which is secreted into the seminal plasma and saturated with spermatozoa just after ejaculation. Several studies have reported that relaxin has important roles in improving motility in human sperm. Thereafter, the growing interest on relaxin has intensified efforts to investigate the role of relaxin in other sperm physiological phenomena like, capacitation, acrosome reaction, and their mediating factors associated with successful fertilization. Therefore, this review aims to provide up-to-date information about the physiological roles of relaxin in sperm motility, capacitation, acrosome reaction, and their mediating factors, such as, utilization of glucose, cholesterol efflux, Ca2+-influx, intracellular cAMP and protein tyrosine phosphorylation. Some studies have shown relaxin to increase the percentage of progressive motility and induce capacitation and acrosome reaction through increasing the utilization of glucose and mediating the cholesterol efflux, Ca2+-influx, intracellular cAMP and protein tyrosine phosphorylation. Thus, the review suggests that the supplementation of relaxin into the capacitating medium may contribute the possible beneficial roles in fresh and cryopreserved spermatozoal prefertilization events.

Keywords: relaxin, physiological roles, prefertilizing activities, spermatozoa

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Authors: JiYoung Park, SueGoo Rhee, HyunAe Woo

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In liver regeneration, quiescent hepatocytes need to be primed to fully respond to growth factors such as hepatocyte growth factor. To understand the priming process, it is necessary to analyze patterns of gene expression that occur during liver regeneration after partial hepatectomy (PHx). Recently, tyrosine phosphorylation of signal transducer and activator of transcription 3 (pYSTAT3) has been shown to play an important role in initiating liver regeneration. In order to evaluate the role of pYSTAT3 on liver regeneration after PHx, we used an intrabody which can selectively inhibit pYSTAT3. In our previous studies, an intrabody had been shown that it bound specifically to the pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells, as well as mouse liver, blocked both accumulation of pYSTAT3 in the nucleus and downstream target of pYSTAT3. In this study, PHx was performed on intrabody-expressing mice and the expression levels of liver regeneration-related genes were analyzed. We also measured liver/body weight ratios and the related cellular signaling pathways were analyzed. Acute phase response genes were reduced in an intrabody-expressing mice during liver regeneration than in control virus-injected mice. However, the time course of liver mass restoration in intrabody-expressing mice was similar to that observed in control virus-injected mice. We also observed that the expression levels of anti-apoptotic genes, such as Bcl2 and Bcl-xL were decreased in intrabody-expressing mice whereas the expression of cell cycle-related genes such as cyclin D1, and c-myc was increased. Liver regeneration after PHx was partially impaired by the selective inhibition of pYSTAT3 with a phosphorylation site-specific intrabody and these results indicated that pYSTAT3 might have limited role in liver mass recovery.

Keywords: STAT3, pYSTAT3, liver regeneration, intrabody

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Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

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Authors: Mohammad Aladwan

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Alzheimer’s disease (AD) is a widespread dementing illness with a complex and poorly understood etiology. An important role in improving our understanding of the AD process is the modeling of disease-associated changes in tau protein phosphorylation, a protein known to mediate events essential to the onset and progression of AD. A main feature of AD is the abnormal phosphorylation of tau protein and the presence of neurofibrillary tangles. In order to evaluate the respective roles of the microtubule-binding region (MTBR) and alternatively spliced exons in the N-terminal projection domains in AD, we have constructed SHSY-5Y cell lines that stably overexpress four different species of tau protein (4R2N, 4R0N, N(E-2), N(E+2)). Since the toxicity and spreading of tau lesions in AD depends on the interactions of tau with other proteins, we have performed a proteomic analysis of exosome-fraction interactomes for cell lysates and media samples that were isolated from SHSY-5Y cell lines. Functional analysis of tau interactomes based on gene ontology (GO) terms was performed using the String 10.5 database program. The highest number of exosomes proteomes and tau associated proteins were found with 4R2N isoform (2771 and 159) in cell lysate and they have a high strength of connectivity (78%) between proteins, while N(E-2) isoform in the media proteomes has the highest number of proteins and tau associated protein (1829 and 205). Moreover, known AD markers were significantly enriched in secreted interactomes relative to lysate interactomes in the SHSY-5Y cells of tau isoforms lacking exons 2 and 3 in the N-terminal. The lack of exon 2 (E-2) from tau protein can be mediated by tau secretion and spreading to different cells. Enriched functions in the secreted E-2 interactome include signaling and developmental pathways that have been linked to a) tau misprocessing and lesion development and b) tau secretion and which, therefore, could play novel roles in AD pathogenesis.

Keywords: Alzheimer's disease, dementia, tau protein, neurodegenration disease

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Authors: Michal Kielbik, Izabela Szulc-Kielbik, Anna Brzostek, Jaroslaw Dziadek, Magdalena Klink

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The goal of many research groups in the world is to find new components that are important for survival of mycobacteria in the host cells. Mycobacterium tuberculosis (Mtb) possesses a number of enzymes degrading cholesterol that are considered to be an important factor for its survival and persistence in host macrophages. One of them - cholesterol oxidase (ChoD), although not being essential for cholesterol degradation, is discussed as a virulence compound, however its involvement in macrophages’ response to Mtb is still not sufficiently determined. The recognition of tubercle bacilli antigens by pathogen recognition receptors is crucial for the initiation of the host innate immune response. An important receptor that has been implicated in the recognition and/or uptake of Mtb is Toll-like receptor type 2 (TLR2). Engagement of TLR2 results in the activation and phosphorylation of intracellular signaling proteins including IRAK-1 and -4, TRAF-6, which in turn leads to the activation of target kinases and transcription factors responsible for bactericidal and pro-inflammatory response of macrophages. The aim of these studies was a detailed clarification of the role of Mtb cholesterol oxidase as a virulence factor affecting the TLR2 signaling pathway in human macrophages. As human macrophages the THP-1 differentiated cells were applied. The virulent wild-type Mtb strain (H37Rv), its mutant lacking a functional copy of gene encoding cholesterol oxidase (∆choD), as well as complimented strain (∆choD–choD) were used. We tested the impact of Mtb strains on the expression of TLR2-depended signaling proteins (mRNA level, cytosolic level and phosphorylation status). The cytokine and bactericidal response of THP-1 derived macrophages infected with Mtb strains in relation to TLR2 signaling pathway dependence was also determined. We found that during the 24-hours of infection process the wild-type and complemented Mtb significantly reduced the cytosolic level and phosphorylation status of IRAK-4 and TRAF-6 proteins in macrophages, that was not observed in the case of ΔchoD mutant. Decreasement of TLR2-dependent signaling proteins, induced by wild-type Mtb, was not dependent on the activity of proteasome. Blocking of TLR2 expression, before infection, effectively prevented the induced by wild-type strain reduction of cytosolic level and phosphorylation of IRAK-4. None of the strains affected the surface expression of TLR2. The mRNA level of IRAK-4 and TRAF-6 genes were significantly increased in macrophages 24 hours post-infection with either of tested strains. However, the impact of wild-type Mtb strain on both examined genes was significantly stronger than its ΔchoD mutant. We also found that wild-type strain stimulated macrophages to release high amount of immunosuppressive IL-10, accompanied by low amount of pro-inflammatory IL-8 and bactericidal nitric oxide in comparison to mutant lacking cholesterol oxidase. The influence of wild-type Mtb on this type of macrophages' response strongly dependent on fully active IRAK-1 and IRAK-4 signaling proteins. In conclusion, Mtb using cholesterol oxidase causes the over-activation of TLR2 signaling proteins leading to the reduction of their cytosolic level and activity resulting in the modulation of macrophages response to allow its intracellular survival. Supported by grant: 2014/15/B/NZ6/01565, National Science Center, Poland

Keywords: Mycobacterium tuberculosis, cholesterol oxidase, macrophages, TLR2-dependent signaling pathway

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Authors: Enjie Xu, Zhen Huang, Kunpeng Zhu, Jianping Hu, Xiaolong Ma, Yongjie Wang, Jiazhuang Zhu, Chunlin Zhang

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Abstract: Hypoxia and dedifferentiation of osteosarcoma (OS) cells leads to poor prognosis. We plan to identify the role of hypoxia on dedifferentiation and the associated signaling pathways. We performed a sphere formation assay and determined spheroid cells as dedifferentiated cells by detecting stem cell-like markers. RNAi assay was used to explore the expression relationship between hypoxia inducible factor 1 subunit alpha (HIF1A) and platelet derived growth factor receptor beta (PDGFRB). We obtained PDGFRB knockdown and overexpression cells through lentiviral infection experiments and the effects of PDGFRB on cytoskeleton rearrangement and cell adhesion were explored by immunocytochemistry. Wound-healing experiments, transwell assays, and animal trials were employed to investigate the effect of PDGFRB on OS metastasis. Dedifferentiated OS cells were found to exhibit high expression of HIF1A and PDGFRB, and HIF1A promoted the expression of PDGFRB, subsequently activated ras homolog family member A (RhoA), and increased the phosphorylation of myosin light chain (MLC). PDGFRB also enhanced the phosphorylation of focal adhesion kinase (FAK). The OS cell morphology and vinculin distribution were altered by PDGFRB. PDGFRB also promoted cell dedifferentiation and had a significant impact on the metastasis of OS cells both in vitro and in vivo. Our results demonstrated that HIF1A up-regulated PDGFRB under hypoxic conditions, and PDGFRB regulated the actin cytoskeleton by activating RhoA and subsequently phosphorylating MLC, thereby promoting OS dedifferentiation and pulmonary metastasis.

Keywords: osteosarcoma, dedifferentiation, metastasis, cytoskeleton rearrangement, PDGFRB, hypoxia

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