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Sample records for inducible stat1 binding

  1. Double-stranded RNA induces biphasic STAT1 phosphorylation by both type I interferon (IFN)-dependent and type I IFN-independent pathways.

    PubMed

    Dempoya, Junichi; Matsumiya, Tomoh; Imaizumi, Tadaatsu; Hayakari, Ryo; Xing, Fei; Yoshida, Hidemi; Okumura, Ken; Satoh, Kei

    2012-12-01

    Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system.

  2. IL-27 Induces Th17 Differentiation in the Absence of STAT1 Signaling.

    PubMed

    Peters, Anneli; Fowler, Kevin D; Chalmin, Fanny; Merkler, Doron; Kuchroo, Vijay K; Pot, Caroline

    2015-11-01

    It is known that differentiation of Th17 cells is promoted by activation of STAT3 and inhibited by activation of STAT1. Although both transcription factors are activated by several cytokines, including IL-6, IL-21, and IL-27, each of these cytokines has a very different effect on Th17 differentiation, ranging from strong induction (IL-6) to strong inhibition (IL-27). To determine the molecular basis for these differences, we measured STAT3 and STAT1 activation profiles for IL-6, IL-21, and IL-27, as well as for cytokine pairs over time. We found that the ratio of activated STAT3/activated STAT1 is crucial in determining whether cytokines promote or inhibit Th17 differentiation. IL-6 and IL-21 induced p-STAT3/p-STAT1 ratios > 1, leading to the promotion of Th17 differentiation, whereas IL-27 or IL-6+IL-27 induced p-STAT3/p-STAT1 ratios < 1, resulting in inhibition of Th17 differentiation. Consistent with these findings, we show that IL-27 induces sufficient p-STAT3 to promote Th17 differentiation in the absence of STAT1. Furthermore, IL-27-induced STAT1-deficient T cells were indistinguishable from bona fide highly proinflammatory Th17 cells because they induced severe experimental autoimmune encephalomyelitis upon adoptive transfer. Our results suggest that the ratio of p-STAT3/p-STAT1 induced by a cytokine or cytokine pairs can be used to predict whether they induce a competent Th17-differentiation program.

  3. A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

    PubMed Central

    2012-01-01

    Background The Signal Transducer and Activator of Transcription 3 (STAT3) is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs), which bind to the DNA Binding Domain (DBD) of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes. Results New dODNs with a hairpin (hpdODNs) were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i) cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii) absence of inhibition of interferon (IFN) γ-dependent cell death, iii) expression of STAT1 targets, and iv) nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1. Conclusions Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes, known to modulate

  4. Mapping of Stat3 serine phosphorylation to a single residue (727) and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3.

    PubMed Central

    Wen, Z; Darnell, J E

    1997-01-01

    During their polypeptide ligand-induced activation Stats (signaltransducers andactivators oftranscription) 1 and 3 acquire, in addition to an obligatory tyrosine phosphorylation, phosphorylation on serine which boosts their transactivating potential [Wen, Z., Zhong, Z. and Darnell, J. E. Jr. (1995) Cell 82, 241-250]. By examining phosphopeptide maps of wild-type and mutant protein we show here that the Stat3 serine phosphorylation, like the Stat1 serine phosphorylation, occurs on a single residue, serine 727. Neither the DNA binding of Stat1 nor Stat3 is demonstrably affected by the presence or absence of the serine phosphorylation. Thus the earlier demonstration that transcription is enhanced by the presence of the serine 727 residue likely occurs after DNA binding. These findings do not agree with earlier claims of excess serine to tyrosine phosphorylation in activated Stats 1 and 3 or to claims of more stable DNA binding of serine phosphorylated Stat dimers. PMID:9153303

  5. Potential role of 8-oxoguanine DNA glycosylase 1 as a STAT1 coactivator in endotoxin-induced inflammatory response.

    PubMed

    Kim, Hong Sook; Kim, Byung-Hak; Jung, Joo Eun; Lee, Chang Seok; Lee, Hyun Gyu; Lee, Jung Weon; Lee, Kun Ho; You, Ho Jin; Chung, Myung-Hee; Ye, Sang-Kyu

    2016-04-01

    Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases. PMID:26496208

  6. Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.

    PubMed

    Takezaki, Shunichiro; Yamada, Masafumi; Kato, Masahiko; Park, Myoung-Ja; Maruyama, Kenichi; Yamazaki, Yasuhiro; Chida, Natsuko; Ohara, Osamu; Kobayashi, Ichiro; Ariga, Tadashi

    2012-08-01

    Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.

  7. Loss of interferon-induced Stat1 phosphorylation in activated T cells.

    PubMed

    Van De Wiele, C Justin; Marino, Julie H; Whetsell, Michael E; Vo, Stephen S; Masengale, Rhonda M; Teague, T Kent

    2004-03-01

    Modulation of cytokine responsiveness following T cell activation represents an important mechanism that shapes the fate of T cells after encounters with antigens. We activated T cells in mice with superantigen and assessed their ability to phosphorylate Stat1 in response to interferon-gamma (IFN-gamma) and IFN-alpha. After 4 h of activation in vivo, T cells became deficient in their ability to phosphorylate Stat1 in response to either cytokine. The loss of IFN sensitivity was accompanied by increased mRNA transcription for multiple suppressors of cytokine signaling (SOCS) genes (SOCS1, SOCS3, and SOCS7). The transcript levels of these SOCS were elevated only during the early hours after activation and were at or below normal levels by 60 h. Likewise, the activation-induced inhibition of IFN-alpha signaling was transient, and sensitivity was restored by 3 days postactivation. The loss of sensitivity to IFN-gamma persisted, however, and was still evident at 3 days. These data suggest that SOCS-independent mechanisms specific for inhibition of IFN-gamma signaling may be present at later stages of the T cell response. The loss of Stat1 signaling may be a factor in differentiation of T cells during and after activation, and it could also represent a protective mechanism against the toxic effects of IFN-gamma during immune responses.

  8. STS-1 promotes IFN-α induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells.

    PubMed

    Dong, Guanjun; You, Ming; Fan, Hongye; Ding, Liang; Sun, Lingyun; Hou, Yayi

    2015-08-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN-α. IFN-α induces autophagy via the JAK1-STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS-1 (suppressor of T-cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS-1 promoted IFN-α-induced autophagy in B cells by enhancing the JAK1-STAT1 signaling activation. STS-1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c-cbl, and subsequently promoted IFN-α-induced phosphorylation of tyrosine kinase 2, leading to JAK1-STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN-α-induced autophagy promoted by STS-1, indicating that STS-1 promotes IFN-α-induced autophagy via the JAK1-STAT1 signaling. Our results demonstrate the importance of STS-1 in regulating IFN-α-induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.

  9. Paeonol attenuates TNBS-induced colitis by inhibiting NF-{kappa}B and STAT1 transactivation

    SciTech Connect

    Ishiguro, Kazuhiro . E-mail: kio@med.nagoya-u.ac.jp; Ando, Takafumi; Maeda, Osamu; Hasegawa, Motofusa; Kadomatsu, Kenji; Ohmiya, Naoki; Niwa, Yasumasa; Xavier, Ramnik; Goto, Hidemi

    2006-11-15

    Paeonol, a major phenolic component of Moutan Cortex, is known to have anti-inflammatory activity. However, the effect of Paeonol on colitis has not been evaluated and the molecular mechanism of its anti-inflammatory action remains unknown. The aim of this study was to determine if Paeonol enema attenuates trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. We also investigated the effects of Paeonol in colon cancer-derived CW-2 cells and T cell leukemia-derived Jurkat cells treated with tumor necrosis factor {alpha} (TNF{alpha}) and/or interferon {gamma} (IFN{gamma}), which play critical roles in TNBS-induced colitis. Paeonol enema attenuated TNBS-induced colitis judging by body weigh reduction, colon length and histological score. Myeloperoxidase activity and inducible nitric oxide synthase (iNOS) production in the colon were also reduced with Paeonol enema. In CW-2 cells, Paeonol inhibited iNOS protein and mRNA expression induced by costimulation of TNF{alpha} and IFN{gamma}. Furthermore, Paeonol reduced TNF{alpha}-induced NF-{kappa}B transactivation and IFN{gamma}-induced STAT1 transactivation in CW-2 cells and also in Jurkat cells. These findings suggest that Paeonol enema may be useful for the treatment of colitis.

  10. Deubiquitinase USP2a Sustains Interferons Antiviral Activity by Restricting Ubiquitination of Activated STAT1 in the Nucleus

    PubMed Central

    Liu, Jin; Yuan, Yukang; Cheng, Qiao; Zuo, Yibo; Qian, Liping; Guo, Tingting; Qian, Guanghui; Li, Lemin; Ge, Jun; Dai, Jianfeng; Xiong, Sidong; Zheng, Hui

    2016-01-01

    STAT1 is a critical transcription factor for regulating host antiviral defenses. STAT1 activation is largely dependent on phosphorylation at tyrosine 701 site of STAT1 (pY701-STAT1). Understanding how pY701-STAT1 is regulated by intracellular signaling remains a major challenge. Here we find that pY701-STAT1 is the major form of ubiquitinated-STAT1 induced by interferons (IFNs). While total STAT1 remains relatively stable during the early stages of IFNs signaling, pY701-STAT1 can be rapidly downregulated by the ubiquitin-proteasome system. Moreover, ubiquitinated pY701-STAT1 is located predominantly in the nucleus, and inhibiting nuclear import of pY701-STAT1 significantly blocks ubiquitination and downregulation of pY701-STAT1. Furthermore, we reveal that the deubiquitinase USP2a translocates into the nucleus and binds to pY701-STAT1, and inhibits K48-linked ubiquitination and degradation of pY701-STAT1. Importantly, USP2a sustains IFNs-induced pY701-STAT1 levels, and enhances all three classes of IFNs- mediated signaling and antiviral activity. To our knowledge, this is the first identified deubiquitinase that targets activated pY701-STAT1. These findings uncover a positive mechanism by which IFNs execute efficient antiviral signaling and function, and may provide potential targets for improving IFNs-based antiviral therapy. PMID:27434509

  11. Activation of JAK2/STAT1-alpha-dependent signaling events during Mycobacterium tuberculosis-induced macrophage apoptosis.

    PubMed

    Rojas, Mauricio; Olivier, Martin; García, Luis F

    2002-01-01

    Induction of apoptosis by Mycobacterium tuberculosis in murine macrophage involves TNF-alpha and nitric oxide (NO) production and caspase cascade activation; however, the intracellular signaling pathways implicated remain to be established. Our results indicate that infection of the B10R murine macrophage line with M. tuberculosis induces apoptosis independent of mycobacterial phagocytosis and that M. tuberculosis induces protein tyrosine kinase (PTK) activity, JAK2/STAT1-alpha phosphorylation, and STAT1-alpha nuclear translocation. Inhibitors of PTK (AG-126), or JAK2 (AG-490) inhibited TNF-alpha and NO production, caspase 1 activation and apoptosis, suggesting that M. tuberculosis-induction of these events depends on JAK2/STAT1-alpha activation. In addition, we have obtained evidence that ManLAM capacity to inhibit M. tuberculosis-induced apoptosis involves the activation of the PTP SHP-1. The finding that M. tuberculosis infection activate JAK2/STAT1-alpha pathway suggests that M. tuberculosis might mimic macrophage-activating stimuli.

  12. Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells

    PubMed Central

    Kang, Ho-Bum; Ahn, Kyung-Seop; Oh, Sei-Ryang; Kim, Jae Wha

    2014-01-01

    The flower buds of Daphne genkwa Sieb. et Zucc. have been used as a traditional Chinese medicine although their functional mechanisms have not been discovered yet. We have studied the potential effects of the plant extracts on natural killer (NK) cell activation, and isolated an active fraction. Genkwadaphnin (GD-1) displayed a potent efficacy to induce IFN-γ transcription in NK cells with concentration- and time-dependent manners. GD-1 treatment triggered the phosphorylation of PKD1, a member of PKC family, MEK and ERK, resulting in IKK activation to induce IκB degradation, and the nuclear localization of p65, an NF-κB subunit, which regulates IFN-γ transcription. GD-1 effect on IFN-γ production was blocked by the addition of Rottlerin, a PKC inhibitor, CID 755673, a PKD inhibitor, or Bay11-7082, an IKKα inhibitor. The nuclear localization of p65 was also inhibited by the kinase inhibitors. Secreted IFN-γ activates STAT1 phosphorylation as autocrine-loops to sustain its secretion. GD-1 induced the phosphorylation of STAT1 probably through the increase of IFN-γ. STAT1 inhibitor also abrogated the sustained IFN-γ secretion. These results suggest that GD-1 is involved in the activation of PKD1 and/or ERK pathway, which activate NK-κB triggering IFN-γ production. As positive feedback loops, secreted IFN-γ activates STAT1 and elongates its production in NK-92 cells. PMID:25517939

  13. Liver X receptor and STAT1 cooperate downstream of Gas6/Mer to induce anti-inflammatory arginase 2 expression in macrophages

    PubMed Central

    Kim, Si-Yoon; Lim, Eun-Jin; Yoon, Young-So; Ahn, Young-Ho; Park, Eun-Mi; Kim, Hee-Sun; Kang, Jihee Lee

    2016-01-01

    Mer signaling increases the transcriptional activity of liver X receptor (LXR) to promote the resolution of acute sterile inflammation. Here, we aimed to understand the pathway downstream of Mer signaling after growth arrest-specific protein 6 (Gas6) treatment that leads to LXR expression and transcriptional activity in mouse bone-marrow derived macrophages (BMDM). Gas6-induced increases in LXRα and LXRβ and expression of their target genes were inhibited in BMDM from STAT1−/− mice or by the STAT1-specific inhibitor fludarabine. Gas6-induced STAT1 phosphorylation, LXR activation, and LXR target gene expression were inhibited in BMDM from Mer−/− mice or by inhibition of PI3K or Akt. Gas6-induced Akt phosphorylation was inhibited in BMDM from STAT1−/− mice or in the presence of fludarabine. Gas6-induced LXR activity was enhanced through an interaction between LXRα and STAT1 on the DNA promoter of Arg2. Additionally, we found that Gas6 inhibited lipopolysaccharide (LPS)-induced nitrite production in a STAT1 and LXR pathway-dependent manner in BMDM. Additionally, Mer-neutralizing antibody reduced LXR and Arg2 expression in lung tissue and enhanced NO production in bronchoalveolar lavage fluid in LPS-induced acute lung injury. Our data suggest the possibility that the Gas6-Mer-PI3K/Akt-STAT1-LXR-Arg2 pathway plays an essential role for resolving inflammatory response in acute lung injury. PMID:27406916

  14. Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

    PubMed

    Strandin, Tomas; Hepojoki, Jussi; Laine, Outi; Mäkelä, Satu; Klingström, Jonas; Lundkvist, Åke; Julkunen, Ilkka; Mustonen, Jukka; Vaheri, Antti

    2016-05-15

    Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

  15. IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

    PubMed Central

    Bardhan, Kankana; Paschall, Amy V.; Yang, Dafeng; Chen, May R.; Simon, Priscilla S.; Bhutia, Yangzom; Martin, Pamela M.; Thangaraju, Muthusamy; Browning, Darren D.; Ganapathy, Vadivel; Heaton, Christopher M.; Gu, Keni; Lee, Jeffrey R.; Liu, Kebin

    2015-01-01

    Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

  16. A time- and dose-dependent STAT1 expression system

    PubMed Central

    Leitner, Nicole R; Strobl, Birgit; Bokor, Marion; Painz, Ronald; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Karaghiosoff, Marina

    2006-01-01

    Background The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. Results The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. Conclusion These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner. PMID:17184522

  17. ROS-dependent Syk and Pyk2-mediated STAT1 activation is required for 15(S)-Hydroxyeicosatetraenoic acid-induced CD36 expression and foam cell formation

    PubMed Central

    Kotla, Sivareddy; Singh, Nikhlesh K.; Traylor, James G.; Orr, A. Wayne; Rao, Gadiparthi N.

    2014-01-01

    15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1/2 (15-LO1/2) metabolite of arachidonic acid (AA), induces CD36 expression through xanthine oxidase and NADPH oxidase-dependent ROS production and Syk and Pyk2-dependent STAT1 activation. In line with these observations, 15(S)-HETE also induced foam cell formation involving ROS, Syk, Pyk2 and STAT1-mediated CD36 expression. In addition, peritoneal macrophages from Western diet-fed ApoE−/− mice exhibited elevated levels of xanthine oxidase and NADPH oxidase activities, ROS production, Syk, Pyk2, and STAT1 phosphorylation and CD36 expression compared to those from ApoE−/−:12/15-LO−/− mice and these events correlated with increased lipid deposits, macrophage content and lesion progression in the aortic roots. Human atherosclerotic arteries also showed increased 15-LO1 expression, STAT1 phosphorylation and CD36 levels as compared to normal arteries. Together, these findings suggest that 12/15-LO metabolites of AA, particularly 12/15(S)-HETE might play a crucial role in atherogenesis by enhancing foam cell formation. PMID:25152235

  18. STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

    PubMed

    Wu, Chengsheng; Molavi, Ommoleila; Zhang, Haifeng; Gupta, Nidhi; Alshareef, Abdulraheem; Bone, Kathleen M; Gopal, Keshav; Wu, Fang; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Lai, Raymond

    2015-07-16

    The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

  19. TNFα and IFNγ Synergistically Enhance Transcriptional Activation of CXCL10 in Human Airway Smooth Muscle Cells via STAT-1, NF-κB, and the Transcriptional Coactivator CREB-binding Protein

    PubMed Central

    Clarke, Deborah L.; Clifford, Rachel L.; Jindarat, Sarawut; Proud, David; Pang, Linhua; Belvisi, Maria; Knox, Alan J.

    2010-01-01

    Asthmatic airway smooth muscle (ASM) expresses interferon-γ-inducible protein-10 (CXCL10), a chemokine known to mediate mast cell migration into ASM bundles that has been reported in the airways of asthmatic patients. CXCL10 is elevated in patients suffering from viral exacerbations of asthma and in patients with chronic obstructive pulmonary disease (COPD), diseases in which corticosteroids are largely ineffective. IFNγ and TNFα synergistically induce CXCL10 release from human ASM cells in a steroid-insensitive manner, via an as yet undefined mechanism. We report that TNFα activates the classical NF-κB (nuclear factor κB) pathway, whereas IFNγ activates JAK2/STAT-1α and that inhibition of the JAK/STAT pathway is more effective in abrogating CXCL10 release than the steroid fluticasone. The synergy observed with TNFα and IFNγ together, however, did not lie at the level of NF-κB activation, STAT-1α phosphorylation, or in vivo binding of these transcription factors to the CXCL10 promoter. Stimulation of human ASM cells with TNFα and IFNγ induced histone H4 but not histone H3 acetylation at the CXCL10 promoter, although no synergism was observed when both cytokines were combined. We show, however, that TNFα and IFNγ exert a synergistic effect on the recruitment of CREB-binding protein (CBP) to the CXCL10, which is accompanied by increased RNA polymerase II. Our results provide evidence that synergism between TNFα and IFNγ lies at the level of coactivator recruitment in human ASM and suggest that inhibition of JAK/STAT signaling may be of therapeutic benefit in steroid-resistant airway disease. PMID:20833730

  20. TNFα and IFNγ synergistically enhance transcriptional activation of CXCL10 in human airway smooth muscle cells via STAT-1, NF-κB, and the transcriptional coactivator CREB-binding protein.

    PubMed

    Clarke, Deborah L; Clifford, Rachel L; Jindarat, Sarawut; Proud, David; Pang, Linhua; Belvisi, Maria; Knox, Alan J

    2010-09-17

    Asthmatic airway smooth muscle (ASM) expresses interferon-γ-inducible protein-10 (CXCL10), a chemokine known to mediate mast cell migration into ASM bundles that has been reported in the airways of asthmatic patients. CXCL10 is elevated in patients suffering from viral exacerbations of asthma and in patients with chronic obstructive pulmonary disease (COPD), diseases in which corticosteroids are largely ineffective. IFNγ and TNFα synergistically induce CXCL10 release from human ASM cells in a steroid-insensitive manner, via an as yet undefined mechanism. We report that TNFα activates the classical NF-κB (nuclear factor κB) pathway, whereas IFNγ activates JAK2/STAT-1α and that inhibition of the JAK/STAT pathway is more effective in abrogating CXCL10 release than the steroid fluticasone. The synergy observed with TNFα and IFNγ together, however, did not lie at the level of NF-κB activation, STAT-1α phosphorylation, or in vivo binding of these transcription factors to the CXCL10 promoter. Stimulation of human ASM cells with TNFα and IFNγ induced histone H4 but not histone H3 acetylation at the CXCL10 promoter, although no synergism was observed when both cytokines were combined. We show, however, that TNFα and IFNγ exert a synergistic effect on the recruitment of CREB-binding protein (CBP) to the CXCL10, which is accompanied by increased RNA polymerase II. Our results provide evidence that synergism between TNFα and IFNγ lies at the level of coactivator recruitment in human ASM and suggest that inhibition of JAK/STAT signaling may be of therapeutic benefit in steroid-resistant airway disease.

  1. STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

    SciTech Connect

    Blank, Viviana C.; Pena, Clara; Roguin, Leonor P.

    2010-02-15

    In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

  2. Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock.

    PubMed

    Lee, Hwi-Ho; Shin, Ji-Sun; Lee, Woo-Seok; Ryu, Byeol; Jang, Dae Sik; Lee, Kyung-Tae

    2016-04-22

    Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1. PMID:26977531

  3. Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock.

    PubMed

    Lee, Hwi-Ho; Shin, Ji-Sun; Lee, Woo-Seok; Ryu, Byeol; Jang, Dae Sik; Lee, Kyung-Tae

    2016-04-22

    Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1.

  4. PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

    PubMed

    Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

    2016-04-01

    Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases.

  5. Phagosomes induced by cytokines function as anti-Listeria vaccines: novel role for functional compartmentalization of STAT-1 protein and cathepsin-D.

    PubMed

    Carrasco-Marín, Eugenio; Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M Jesús; Ruiz-Sáez, Marta; Madrazo-Toca, Fidel; Hölscher, Christoph; Alvarez-Dominguez, Carmen

    2012-04-27

    Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.

  6. Shp-2 contributes to anti-RSV activity in human pulmonary alveolar epithelial cells by interfering with the IFN-α-induced Jak/Stat1 pathway

    PubMed Central

    Wang, Saisai; Zheng, Gang; Zhao, Lifang; Xu, Feng; Qian, Jing

    2015-01-01

    Src homology phosphotyrosyl phosphatase 2 (Shp-2) is a ubiquitously expressed protein that is involved in a variety of cellular processes, including antiviral interferon signalling pathways. In this study, we investigated the role of Shp-2 in the host cell interactions of human respiratory syncytial virus (RSV). We report significant changes in the expression of Shp-2 in human pulmonary alveolar epithelial cells (A549) upon RSV infection. We also report that blocking Shp-2 does not affect viral replication or virus-induced interferon-alpha (IFN-α) production. Interestingly, whereas A549 cells were activated by IFN-α, the blocking of Shp-2 resulted in increased viral replication that was associated with the reduced expression of the IFN-stimulated genes of 2′,5′-oligoadenylate synthetases and Mx1, and the concomitant inhibition of Stat1 tyrosine phosphorylation. Our findings suggest that Shp-2 contributes to the control of RSV replication and progeny production in pulmonary alveolar epithelial cells by interfering with IFN-α-induced Jak/Stat1 pathway activation rather than by affecting the production of IFN-α itself. PMID:26119280

  7. Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis

    PubMed Central

    Sampaio, Elizabeth P.; Hsu, Amy P.; Pechacek, Joseph; Bax, Hannelore I.; Dias, Dalton L.; Paulson, Michelle L.; Chandrasekaran, Prabha; Rosen, Lindsey B.; Carvalho, Daniel S.; Ding, Li; Vinh, Donald C.; Browne, Sarah K.; Datta, Shrimati; Milner, Joshua D.; Kuhns, Douglas B.; Long Priel, Debra A.; Sadat, Mohammed A.; Shiloh, Michael; De Marco, Brendan; Alvares, Michael; Gillman, Jason W.; Ramarathnam, Vivek; de la Morena, Maite; Bezrodnik, Liliana; Moreira, Ileana; Uzel, Gulbu; Johnson, Daniel; Spalding, Christine; Zerbe, Christa S.; Wiley, Henry; Greenberg, David E.; Hoover, Susan E.; Rosenzweig, Sergio D.; Galgiani, John N.; Holland, Steven M.

    2013-01-01

    Background Impaired signaling in the IFN-γ/IL-12 pathway causes susceptibility to severe disseminated infections with mycobacteria and dimorphic yeasts. Dominant gain-of-function mutations in signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis. Objective We sought to identify the molecular defect in patients with disseminated dimorphic yeast infections. Methods PBMCs, EBV-transformed B cells, and transfected U3A cell lines were studied for IFN-γ/IL-12 pathway function. STAT1 was sequenced in probands and available relatives. Interferon-induced STAT1 phosphorylation, transcriptional responses, protein-protein interactions, target gene activation, and function were investigated. Results We identified 5 patients with disseminated Coccidioides immitis or Histoplasma capsulatum with heterozygous missense mutations in the STAT1 coiled-coil or DNA-binding domains. These are dominant gain-of-function mutations causing enhanced STAT1 phosphorylation, delayed dephosphorylation, enhanced DNA binding and transactivation, and enhanced interaction with protein inhibitor of activated STAT1. The mutations caused enhanced IFN-γ–induced gene expression, but we found impaired responses to IFN-γ restimulation. Conclusion Gain-of-function mutations in STAT1 predispose to invasive, severe, disseminated dimorphic yeast infections, likely through aberrant regulation of IFN-γ–mediated inflammation. PMID:23541320

  8. JAK-STAT1/3-induced expression of signal sequence-encoding proopiomelanocortin mRNA in lymphocytes reduces inflammatory pain in rats

    PubMed Central

    2012-01-01

    Background Proopiomelanocortin (POMC)-derived beta-endorphin1-31 from immune cells can inhibit inflammatory pain. Here we investigated cytokine signaling pathways regulating POMC gene expression and beta-endorphin production in lymphocytes to augment such analgesic effects. Results Interleukin-4 dose-dependently elevated POMC mRNA expression in naïve lymph node-derived cells in vitro, as determined by real-time PCR. This effect was neutralized by janus kinase (JAK) inhibitors. Transfection of Signal Transducer and Activator of Transcription (STAT) 1/3 but not of STAT6 decoy oligonucleotides abolished interleukin-4 induced POMC gene expression. STAT3 was phosphorylated in in vitro interleukin-4 stimulated lymphocytes and in lymph nodes draining inflamed paws in vivo. Cellular beta-endorphin increased after combined stimulation with interleukin-4 and concanavalin A. Consistently, in vivo reduction of inflammatory pain by passively transferred T cells improved significantly when donor cells were pretreated with interleukin-4 plus concanavalin A. This effect was blocked by naloxone-methiodide. Conclusion Interleukin-4 can amplify endogenous opioid peptide expression mediated by JAK-STAT1/3 activation in mitogen-activated lymphocytes. Transfer of these cells leads to inhibition of inflammatory pain via activation of peripheral opioid receptors. PMID:23146666

  9. Biodegradable chitosan microparticles induce delayed STAT-1 activation and lead to distinct cytokine responses in differentially polarized human macrophages in vitro.

    PubMed

    Fong, David; Ariganello, Marianne B; Girard-Lauzière, Joël; Hoemann, Caroline D

    2015-01-01

    Current data suggest that chitosan activates wound macrophages to release endogenous factors that guide mesenchymal stem cell (MSC) to bone fractures. We tested the hypothesis that chitosan, a polymer containing glucosamine and N-acetyl glucosamine, stimulates macrophages in different polarization states to release functional MSC chemokines and mainly anabolic factors. Low-serum conditioned medium was collected from M0, M1 and M2a U937 macrophages previously differentiated with phorbol myristate acetate (PMA) and exposed or not for 24h to chitosan microparticles (80% degree of deacetylation, DDA, 130kDa). Chitosan particles were highly phagocytosed. Chitosan enhanced anabolic factor release from M0 and M2a macrophages (MCP-1, IP-10, MIP-1beta, IL-1ra, IL-10, PDGF), and IL-1beta release, with 25- to 400-fold excess IL-1ra over IL-1beta. In M1 macrophages, chitosan enhanced IL-1beta without enhancing or suppressing inflammatory factor release (IL-6, IP-10, IL-8). M0 and M2a macrophages, with or without chitosan stimulation, produced conditioned medium that promoted 2-fold more MSC chemotaxis than low-serum control medium, while M1-conditioned medium failed to induce MSC chemotaxis. Acetylated chitosan induced U937 macrophages to release IL-1ra without STAT-6 activation, and also induced a delayed STAT-1 activation/IP-10 release response that was not observed using non-biodegradable chitosan (98% DDA, 130kDa). In primary human macrophages, acetylated chitosan enhanced IL-1ra release without inducing IL-1beta, and required PMA priming to elicit STAT-1 activation and IP-10 release. We conclude that biodegradable chitosan particles enhance M0 and M2a macrophage anabolic responses independent of the IL4/STAT-6 axis, by inducing excess IL-1ra over IL-1beta and more chemokine release, without altering their inherent capacity to attract MSCs. PMID:25449925

  10. CDK8 kinase phosphorylates transcription factor STAT1 to selectively regulate the interferon response.

    PubMed

    Bancerek, Joanna; Poss, Zachary C; Steinparzer, Iris; Sedlyarov, Vitaly; Pfaffenwimmer, Thaddäus; Mikulic, Ivana; Dölken, Lars; Strobl, Birgit; Müller, Mathias; Taatjes, Dylan J; Kovarik, Pavel

    2013-02-21

    Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.

  11. Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

    SciTech Connect

    Hui Zhouguang; Tretiakova, Maria; Zhang Zhongfa; Li Yan; Wang Xiaozhen; Zhu, Julie Xiaohong; Gao Yuanhong; Mai Weiyuan; Furge, Kyle; Qian Chaonan; Amato, Robert; Butler, E. Brian

    2009-01-01

    Purpose: Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. Methods and Materials: The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. Results: STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10{sup -8} for clear cell; and p = 3.6 x 10{sup -4} for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. Conclusion: This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

  12. Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC).

    PubMed

    Zheng, Jie; van de Veerdonk, Frank L; Crossland, Katherine L; Smeekens, Sanne P; Chan, Chun M; Al Shehri, Tariq; Abinun, Mario; Gennery, Andrew R; Mann, Jelena; Lendrem, Dennis W; Netea, Mihai G; Rowan, Andrew D; Lilic, Desa

    2015-10-01

    Signal transducer and activator of transcription 3 (STAT3) triggered production of Th-17 cytokines mediates protective immunity against fungi. Mutations affecting the STAT3/interleukin 17 (IL-17) pathway cause selective susceptibility to fungal (Candida) infections, a hallmark of chronic mucocutaneous candidiasis (CMC). In patients with autosomal dominant CMC, we and others previously reported defective Th17 responses and underlying gain-of-function (GOF) STAT1 mutations, but how this affects STAT3 function leading to decreased IL-17 is unclear. We also assessed how GOF-STAT1 mutations affect STAT3 activation, DNA binding, gene expression, cytokine production, and epigenetic modifications. We excluded impaired STAT3 phosphorylation, nuclear translocation, and sequestration of STAT3 into STAT1/STAT3 heterodimers and confirm significantly reduced transcription of STAT3-inducible genes (RORC/IL-17/IL-22/IL-10/c-Fos/SOCS3/c-Myc) as likely underlying mechanism. STAT binding to the high affinity sis-inducible element was intact but binding to an endogenous STAT3 DNA target was impaired. Reduced STAT3-dependent gene transcription was reversed by inhibiting STAT1 activation with fludarabine or enhancing histone, but not STAT1 or STAT3 acetylation with histone deacetylase (HDAC) inhibitors trichostatin A or ITF2357. Silencing HDAC1, HDAC2, and HDAC3 indicated a role for HDAC1 and 2. Reduced STAT3-dependent gene transcription underlies low Th-17 responses in GOF-STAT1 CMC, which can be reversed by inhibiting acetylation, offering novel targets for future therapies.

  13. The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii

    PubMed Central

    Beiting, Daniel P.; Hidano, Shinya; Baggs, Julie E.; Geskes, Jeanne M.; Fang, Qun; Wherry, E. John; Hunter, Christopher A.; Roos, David S.; Cherry, Sara

    2015-01-01

    The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host–pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such “modifiers.” PMID:26196739

  14. Quercetin protects mouse liver against nickel-induced DNA methylation and inflammation associated with the Nrf2/HO-1 and p38/STAT1/NF-κB pathway.

    PubMed

    Liu, Chan-Min; Ma, Jie-Qiong; Xie, Wan-Ru; Liu, Si-Si; Feng, Zhao-Jun; Zheng, Gui-Hong; Wang, Ai-Min

    2015-08-01

    Quercetin (QE), a natural flavonoid, has been reported to have many benefits and medicinal properties. However, its protective effects against nickel (Ni) induced injury in liver have not been clarified. The aim of the present study was to investigate the effects of quercetin on hepatic DNA methylation and inflammation in mice exposed to nickel. ICR mice were exposed to nickel sulfate with or without quercetin co-administration for 20 days. Our results showed that quercetin administration significantly inhibited nickel-induced liver injury, which was indicated by diagnostic indicators. In exploring the underlying mechanisms of quercetin action, we found that quercetin decreased total DNA methyltransferases (DNMTs) activity and DNA methylation level of the NF-E2 related factor 2 (Nrf2) DNA in livers of nickel-treated mice. Quercetin also induced Nrf2 nuclear translocation and heme oxygenase-1 (HO-1) activity. Moreover, quercetin decreased production of pro-inflammatory markers including TNF-α, IL-1β and iNOS. Quercetin significantly inhibited the p38 and signal transducer and activator of transcription 1 (STAT1) activation, which in turn inactivated NF-κB and the inflammatory cytokines in livers of the nickel-treated mice. In conclusion, these results suggested that the inhibition of nickel-induced inflammation by quercetin is associated with its ability to modulate Nrf2/HO-1 and p38/STAT1/NF-κB signaling pathway.

  15. Alantolactone from Saussurea lappa Exerts Antiinflammatory Effects by Inhibiting Chemokine Production and STAT1 Phosphorylation in TNF-α and IFN-γ-induced in HaCaT cells.

    PubMed

    Lim, Hye-Sun; Jin, Sung-Eun; Kim, Ohn-Soon; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

    2015-07-01

    Skin inflammation is the most common condition seen in dermatology practice and can be caused by various allergic reactions and certain toxins or chemicals. In the present study, we investigated the antiinflammatory effects of Saussurea lappa, a medicinal herb, and its marker compounds alantolactone, caryophyllene, costic acid, costunolide, and dehydrocostuslactone in the HaCaT human keratinocyte cell line. HaCaT cells were stimulated with tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), and treated with S. lappa or each of five marker compounds. Chemokine production and expression were analyzed by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Phosphorylation of signal transducer and activator of transcription (STAT) 1 was determined by immunoblotting. Stimulation with TNF-α and IFN-γ significantly increased the production of the following chemokines: thymus-regulated and activation-regulated chemokine (TARC): regulated on activation, normal T-cell expressed and secreted (RANTES): macrophage-derived chemokine (MDC): and interleukin-8 (IL-8). By contrast, S. lappa and the five marker compounds significantly reduced the production of these chemokines by TNF-α and IFN-γ-treated cells. S. lappa and alantolactone suppressed the TNF-α and IFN-γ-stimulated increase in the phosphorylation of STAT1. Our results demonstrate that alantolactone from S. lappa suppresses TNF-α and IFN-γ-induced production of RANTES and IL-8 by blocking STAT1 phosphorylation in HaCaT cells. PMID:25881570

  16. The role of stat1b in zebrafish hematopoiesis

    PubMed Central

    Song, Hao; Yan, Yi-lin; Titus, Tom; He, Xinjun; Postlethwait, John H.

    2011-01-01

    STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by subfunctionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development. PMID:21914475

  17. Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Barchowsky, Aaron

    2009-01-01

    Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

  18. hCAF1/CNOT7 regulates interferon signalling by targeting STAT1

    PubMed Central

    Chapat, Clément; Kolytcheff, Chloé; Le Romancer, Muriel; Auboeuf, Didier; De La Grange, Pierre; Chettab, Kamel; Sentis, Stéphanie; Corbo, Laura

    2013-01-01

    Stringent regulation of the interferon (IFN) signalling pathway is essential for maintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. In resting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumour escape. PMID:23386060

  19. Newly synthesized 'hidabeni' chalcone derivatives potently suppress LPS-induced NO production via inhibition of STAT1, but not NF-κB, JNK, and p38, pathways in microglia.

    PubMed

    Hara, Hirokazu; Ikeda, Ryoko; Ninomiya, Masayuki; Kamiya, Tetsuro; Koketsu, Mamoru; Adachi, Tetsuo

    2014-01-01

    Chalcones are open-chain flavonoids that are biosynthesized in various plants. Some of them possess anti-inflammatory activity. We previously found that chalcone glycosides from Brassica rapa L. 'hidabeni' suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) production in rat microglia highly aggressively proliferating immortalized (HAPI) cells. In this study, to explore chalcone derivatives with potent NO inhibitory activity, we synthesized ten compounds based on 'hidabeni' chalcone and examined their effects on LPS-triggered inducible NO synthase (iNOS) expression and NO production. Compounds C4 and C10 potently inhibited NO production (IC50: 4.19, 2.88 µM, respectively). C4 and C10 suppressed LPS-induced iNOS expression via the inhibition of the signal transduction and activator of transcription 1 (STAT1), but not nuclear factor-kappa B (NF-κB), c-Jun N terminal kinase (JNK), and p38, pathways. C10, but not C4, inhibited activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. C4 and C10 also suppressed LPS-induced expression of interferon regulatory factor 1 (IRF-1), which is an important transcription factor involved in iNOS expression. Our findings indicate that these chalcone derivatives are candidate compounds for preventing microglia-mediated neuroinflammation.

  20. Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling.

    PubMed

    Jackson, Sharon H; Yu, Cheng-Rong; Mahdi, Rashid M; Ebong, Samuel; Egwuagu, Charles E

    2004-02-15

    In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.

  1. Phosphorylation of IRF8 in a pre-associated complex with Spi-1/PU.1 and non-phosphorylated Stat1 is critical for LPS induction of the IL1B gene.

    PubMed

    Unlu, Sebnem; Kumar, Arvind; Waterman, Wayne R; Tsukada, Junichi; Wang, Kent Z Q; Galson, Deborah L; Auron, Philip E

    2007-07-01

    Rapid induction of transcription is known to be mediated by factors which bind DNA following post-translational modification. We report here that non-tyrosine phosphorylated (NTP)-Stat1 is involved in a cooperative interaction with Spi-1/PU.1 and IRF8 to form a pre-associated, poised complex for IL1B gene induction. A double point mutation at a putative STAT binding site, which overlaps this composite Spi-1 x IRF8 site located in the LPS and IL-1 response element (LILRE), inhibited human IL1B LPS-dependent reporter activity to about 10 percent of the control wild type vector. Chromatin immunoprecipitation revealed stimulation-independent constitutive binding of IRF8, Spi-1 and NTP-Stat1 at the LILRE, while binding of C/EBP beta was activated at an adjacent C/EBP beta site after LPS stimulation. In contrast to Stat1, IRF8 was tyrosine phosphorylated following LPS treatment. Supporting the involvement of NTP-Stat1, LPS-induced IL1B reporter activity in monocytes was enhanced by ectopic expression of NTP-Stat1 Y701F. In contrast, co-expression of a Y211F IRF8 mutein functioned as a dominant-negative inhibitor of LPS-induced IL1B reporter activity. In vitro DNA binding using extracts from LPS-treated monocytes confirmed that the LILRE enhancer constitutively binds a trimolecular complex containing IRF8, Spi-1 and NTP-Stat1. Binding studies using in vitro-expressed proteins revealed that NTP-Stat1 enhanced the binding of Spi-1 and IRF8 to the LILRE. Co-expression of TRAF6, an LPS surrogate, with Spi-1 and IRF8 enhanced IL1B reporter activity in HEK293R cells, which was dramatically reduced when Y211F IRF8 was co-expressed. These results suggest that the rapid transcriptional induction of an important inflammatory gene is dependent upon constitutive cooperative binding of a Spi-1 x IRF8 x NTP-Stat1 complex to the LILRE, which primes the gene for immediate induction following IRF8 phosphorylation. Phosphorylation of chromatin pre-associated factors like IRF8 may be an

  2. Stat1 negatively regulates immune-mediated injury with Anaplasma phagocytophilum infection1

    PubMed Central

    Choi, Kyoung-Seong; Scorpio, Diana G.; Dumler, J. Stephen

    2014-01-01

    Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium, Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFNγ production through the signal transducer and activation of transcription 1 (Stat1) signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knock out (KO) compared to wild type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated pro-inflammatory cytokine/chemokine responses in serum. CD4+ and CD8+ T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type (WT) mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocytes/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of iNOS in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection. PMID:25305312

  3. Regulation of Stat1 protein expression by phenylalanine 172 in the coiled-coil domain

    SciTech Connect

    Hoshino, Akemi; Fleur, Shella Saint; Fujii, Hodaka . E-mail: hodaka@med.nyu.edu

    2006-08-04

    Stat1 plays an essential role in signal transduction and gene expression of various cytokines including interferons (IFNs). Although the mechanism of cytokine-induced activation of Stat1 and transcriptional regulation of Stat1 gene expression have been established, post-transcriptional regulation of Stat1 protein expression is not fully understood. Here, we report identification of a mutant of Stat1 that has decreased expression levels by using inducible translocation trap (ITT), a reporter gene-based detection system of nuclear translocation. The substitution of serine for phenylalanine 172 (F172S) in the coiled-coil domain causes marked decrease in Stat1 protein expression in various cell lines without decreasing its mRNA levels. Our results suggest that the decrease is caused by translational/post-translational mechanisms independent of proteasome machinery. These results suggest a novel potential mechanism of determination of specificity of Stat proteins and showed that the ITT system is a powerful technique to identify mutants of nuclear translocating signal transducers.

  4. The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes.

    PubMed

    Sisler, Jennifer D; Morgan, Magdalena; Raje, Vidisha; Grande, Rebecca C; Derecka, Marta; Meier, Jeremy; Cantwell, Marc; Szczepanek, Karol; Korzun, William J; Lesnefsky, Edward J; Harris, Thurl E; Croniger, Colleen M; Larner, Andrew C

    2015-01-01

    The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice.

  5. The Signal Transducer and Activator of Transcription 1 (STAT1) Inhibits Mitochondrial Biogenesis in Liver and Fatty Acid Oxidation in Adipocytes

    PubMed Central

    Sisler, Jennifer D.; Morgan, Magdalena; Raje, Vidisha; Grande, Rebecca C.; Derecka, Marta; Meier, Jeremy; Cantwell, Marc; Szczepanek, Karol; Korzun, William J.; Lesnefsky, Edward J.; Harris, Thurl E.; Croniger, Colleen M.; Larner, Andrew C.

    2015-01-01

    The transcription factor STAT1 plays a central role in orchestrating responses to various pathogens by activating the transcription of nuclear-encoded genes that mediate the antiviral, the antigrowth, and immune surveillance effects of interferons and other cytokines. In addition to regulating gene expression, we report that STAT1-/- mice display increased energy expenditure and paradoxically decreased release of triglycerides from white adipose tissue (WAT). Liver mitochondria from STAT1-/- mice show both defects in coupling of the electron transport chain (ETC) and increased numbers of mitochondria. Consistent with elevated numbers of mitochondria, STAT1-/- mice expressed increased amounts of PGC1α, a master regulator of mitochondrial biogenesis. STAT1 binds to the PGC1α promoter in fed mice but not in fasted animals, suggesting that STAT1 inhibited transcription of PGC1α. Since STAT1-/- mice utilized more lipids we examined white adipose tissue (WAT) stores. Contrary to expectations, fasted STAT1-/- mice did not lose lipid from WAT. β-adrenergic stimulation of glycerol release from isolated STAT1-/- WAT was decreased, while activation of hormone sensitive lipase was not changed. These findings suggest that STAT1-/- adipose tissue does not release glycerol and that free fatty acids (FFA) re-esterify back to triglycerides, thus maintaining fat mass in fasted STAT1-/- mice. PMID:26689548

  6. Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling

    PubMed Central

    Qiu, Xusheng; Fu, Qiang; Meng, Chunchun; Yu, Shengqing; Zhan, Yuan; Dong, Luna; Song, Cuiping; Sun, Yingjie; Tan, Lei; Hu, Shunlin; Wang, Xiaoquan; Liu, Xiaowen; Peng, Daxin; Liu, Xiufan; Ding, Chan

    2016-01-01

    Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN. PMID:26859759

  7. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  8. Absence of STAT1 in donor-derived plasmacytoid dendritic cells results in increased STAT3 and attenuates murine GVHD

    PubMed Central

    Nasholm, Nicole M.; Chien, Christopher D.; Larabee, Shannon M.; Qin, Haiying; Song, Young K.; Klover, Peter J.; Hennighausen, Lothar; Khan, Javed; Fry, Terry J.

    2014-01-01

    Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. Transplantation of STAT1−/− bone marrow (BM) prevented GVHD induced by STAT1+/+ T cells, leading to expansion of B220+ cells and regulatory T cells. STAT1−/− BM also preserved GVT activity and enhanced overall survival of tumor-challenged mice in the setting of GVHD. Furthermore, recipients of allogeneic STAT1−/− BM demonstrated increased CD9−Siglec Hhi plasmacytoid dendritic cells (pDCs), and depletion of pDCs after STAT1−/− BM transplantation prevented GVHD resistance. STAT1−/− pDCs were found to produce decreased free radicals, IFNα, and interleukin (IL)-12, and increased IL-10. Additionally, STAT1−/− pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9−/− BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1−/− pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis. PMID:25079358

  9. Two Domains of the V Protein of Virulent Canine Distemper Virus Selectively Inhibit STAT1 and STAT2 Nuclear Import▿

    PubMed Central

    Röthlisberger, Anne; Wiener, Dominique; Schweizer, Matthias; Peterhans, Ernst; Zurbriggen, Andreas; Plattet, Philippe

    2010-01-01

    Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-α/β)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-α/β-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-α/β-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-α/β-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-α/β-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-α/β-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-α/β evasion among the morbilliviruses. PMID:20427537

  10. STAT2 Is a Pervasive Cytokine Regulator due to Its Inhibition of STAT1 in Multiple Signaling Pathways

    PubMed Central

    Ho, Johnathan; Pelzel, Christin; Begitt, Andreas; Mee, Maureen; Elsheikha, Hany M.; Scott, David J.; Vinkemeier, Uwe

    2016-01-01

    STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein. PMID:27780205

  11. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

    PubMed Central

    Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, Mauro

    2016-01-01

    The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. PMID:27437406

  12. STAT1 Signaling within Macrophages Is Required for Antifungal Activity against Cryptococcus neoformans

    PubMed Central

    Leopold Wager, Chrissy M.; Hole, Camaron R.; Wozniak, Karen L.; Olszewski, Michal A.; Mueller, Mathias

    2015-01-01

    Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO. PMID:26351277

  13. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria.

    PubMed

    Schiavano, Giuditta Fiorella; Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, Mauro

    2016-01-01

    The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. PMID:27437406

  14. Bcl6 promotes osteoblastogenesis through Stat1 inhibition

    SciTech Connect

    Fujie, Atsuhiro; Funayama, Atsushi; Miyauchi, Yoshiteru; Sato, Yuiko; Kobayashi, Tami; Kanagawa, Hiroya; Katsuyama, Eri; Hao, Wu; Tando, Toshimi; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Kanaji, Arihiko; Morioka, Hideo; Matsumoto, Morio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-02-13

    Bone mass is tightly controlled by a balance between osteoclast and osteoblast activities. Although these cell types mature via different pathways, some factors reportedly regulate differentiation of both. Here, in a search for factors governing osteoblastogenesis but also expressed in osteoclasts to control both cell types by one molecule, we identified B cell lymphoma 6 (Bcl6) as one of those factors and show that it promotes osteoblast differentiation. Bcl6 was previously shown to negatively regulate osteoclastogenesis. We report that lack of Bcl6 results in significant inhibition of osteoblastogensis in vivo and in vitro and in defects in secondary ossification center formation in vivo. Signal transducer and activator of transcription 1 (Stat1) reportedly attenuates osteoblast differentiation by inhibiting nuclear translocation of runt-related transcription factor 2 (Runx2), which is essential for osteoblast differentiation. We found that lack of Bcl6 resulted in significant elevation of Stat1 mRNA and protein expression in osteoblasts and showed that Stat1 is a direct target of Bcl6 using a chromatin immune-precipitation assay. Mice lacking both Bcl6 and Stat1 (DKO) exhibited significant rescue of bone mass and osteoblastic parameters as well as partial rescue of secondary ossification center formation compared with Bcl6-deficient mice in vivo. Altered osteoblastogenesis in Bcl6-deficient cells was also restored in DKO in vitro. Thus, Bcl6 plays crucial roles in regulating both osteoblast activation and osteoclast inhibition. - Highlights: • Bcl6 is required for osteoblast differentiation. • Bcl6{sup −/−} mice exhibited altered osteoblastogenesis and reduced bone mass in vivo and in vitro. • We identified Stat1 as a direct target of Bcl6 in osteoblasts. • Bcl6 and Stat1 doubly deficient mice exhibited rescued bone phenotypes compared with Bcl6{sup −/−} mice.

  15. IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche.

    PubMed

    Pereira, Leticia; Medina, Rebeca; Baena, Miguel; Planas, Anna M; Pozas, Esther

    2015-01-01

    The adult subventricular zone (SVZ) is the main neurogenic niche in normal adult brains of mice and rats. Interferon gamma (IFNγ) has somewhat controversially been associated with SVZ progenitor proliferation and neurogenesis. The in vivo involvement of IFNγ in the physiology of the adult SVZ niche is not fully understood and its intracellular mediators are unknown. Here we show that IFNγ, through activation of its canonical signal transducer and activator of transcription 1 (STAT1) pathway, acts specifically on Nestin+ progenitors by decreasing both progenitor proliferation and the number of cycling cells. In addition, IFNγ increases the number of neuroblasts generated without shifting glial fate determination. The final result is deficient recruitment of newborn neurons to the olfactory bulb (OB), indicating that IFNγ-induced stimulation of neuronal differentiation does not compensate for its antiproliferative effect. We conclude that IFNγ signaling via STAT1 in the SVZ acts dually as an antiproliferative and proneurogenic factor, and thereby regulates neurogenesis in normal adult brains.

  16. Response to interferons and antibacterial innate immunity in the absence of tyrosine-phosphorylated STAT1.

    PubMed

    Majoros, Andrea; Platanitis, Ekaterini; Szappanos, Daniel; Cheon, HyeonJoo; Vogl, Claus; Shukla, Priyank; Stark, George R; Sexl, Veronika; Schreiber, Robert; Schindler, Christian; Müller, Mathias; Decker, Thomas

    2016-03-01

    Signal transducer and activator of transcription 1 (STAT1) plays a pivotal role in the innate immune system by directing the transcriptional response to interferons (IFNs). STAT1 is activated by Janus kinase (JAK)-mediated phosphorylation of Y701. To determine whether STAT1 contributes to cellular responses without this phosphorylation event, we generated mice with Y701 mutated to a phenylalanine (Stat1(Y701F)). We show that heterozygous mice do not exhibit a dominant-negative phenotype. Homozygous Stat1(Y701F) mice show a profound reduction in Stat1 expression, highlighting an important role for basal IFN-dependent signaling. The rapid transcriptional response to type I IFN (IFN-I) and type II IFN (IFNγ) was absent in Stat1(Y701F) cells. Intriguingly, STAT1Y701F suppresses the delayed expression of IFN-I-stimulated genes (ISG) observed in Stat1(-/-) cells, mediated by the STAT2/IRF9 complex. Thus, Stat1(Y701F) macrophages are more susceptible to Legionella pneumophila infection than Stat1(-/-) macrophages. Listeria monocytogenes grew less robustly in Stat1(Y701F) macrophages and mice compared to Stat1(-/-) counterparts, but STAT1Y701F is not sufficient to rescue the animals. Our studies are consistent with a potential contribution of Y701-unphosphorylated STAT1 to innate antibacterial immunity. PMID:26882544

  17. Interferon alpha-armed nanoparticles trigger rapid and sustained STAT1-dependent anti-viral cellular responses.

    PubMed

    Pollok, Sibyll; Ginter, Torsten; Günzel, Katharina; Pieper, Jana; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Krämer, Oliver H

    2013-04-01

    Interferon-α (IFNα) has enormous potential for anti-proliferative and anti-viral treatments. However, clinical success is still hampered due to its limited bioavailability and thus, lack of sustained modulation of disease-relevant protective programs. Consequently, we here examined whether IFNα immobilized on nanoscale ferromagnetic R-Chitosan carriers is capable of inducing rapid and sustained activation of STAT1 signaling. We report the spontaneous formation of a stable nanoparticle-IFNα protein corona, which was exploited to generate IFNα-loaded spheres, obviating the need to specifically couple the cytokine to the nanoparticles (NPs). Notably, comprehensive experimental approaches ensure that formation of the IFNα NP-corona does not affect the biological activity of the cytokine, as STAT1 signaling was efficiently activated. Employing human prostate cancer and melanoma cell models, we found that the intensity and duration of STAT1 phosphorylation as well as the downstream activation of pathobiologically relevant genes were dose and particle dependent. In comparison with free IFNα, IFNα-loaded spheres resulted in a more sustained biologically relevant STAT1 activation, demonstrated also by conferring innate cellular immunity against vesicular stomatitis virus (VSV) infection. For one, our study demonstrates the advantages of biodegradable IFNα-coated R-Chitosan NPs for controlled cytokine release, and thereby improved therapy. Second, we reveal that the permanent presence of IFNα and not just the initial STAT1 phosphorylation ensures sustained IFNα-dependent signaling. PMID:23333460

  18. Molecular characterization of RIG-I, STAT-1 and IFN-beta in the horseshoe bat.

    PubMed

    Li, Jinju; Zhang, Guangxu; Cheng, Dalong; Ren, Hua; Qian, Min; Du, Bing

    2015-04-25

    Wild Chinese horseshoe bats have been proven to be natural reservoirs of SARS-like coronaviruses. However, the molecular characterization of key proteins in bats still needs to be explored further. In this study, we used cloning and bioinformatics to analyze the sequence of RIG-I, STAT-1 and IFN-β in the immortalized cell lines from Rhinolophus affinis and Rhinolophus sinicus. Then, we treated different bat cells, mouse embryonic fibroblasts (MEF) and splenocytes with polyinosinic-polycytidylic acid (polyI:C) and vesicular stomatitis virus (VSV) to assess and compare antiviral immune responses between bats and mice. Our results demonstrated that bat RIG-I, STAT-1 and IFN-β showed close homology with human, mouse, pig and rhesus monkey. RIG-I and STAT-1 were both highly expressed in bat spleen. Furthermore, IFN-β was induced by polyI:C and VSV in both bat and mouse cells. These findings have provided new insight into the potential characteristics of the bat innate immune system against viral infection. PMID:25680291

  19. TYK2-STAT1-BCL2 Pathway Dependence in T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Sanda, Takaomi; Tyner, Jeffrey W.; Gutierrez, Alejandro; Ngo, Vu N.; Glover, Jason; Chang, Bill H.; Yost, Arla; Ma, Wenxue; Fleischman, Angela G.; Zhou, Wenjun; Yang, Yandan; Kleppe, Maria; Ahn, Yebin; Tatarek, Jessica; Kelliher, Michelle A.; Neuberg, Donna S.; Levine, Ross L.; Moriggl, Richard; Müller, Mathias; Gray, Nathanael S.; Jamieson, Catriona H. M.; Weng, Andrew P.; Staudt, Louis M.; Druker, Brian J.; Look, A. Thomas

    2013-01-01

    Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway. PMID:23471820

  20. Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

    PubMed Central

    2010-01-01

    Background Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant. Results VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2. Conclusions Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN

  1. Radiation abolishes inducer binding to lactose repressor.

    PubMed

    Gillard, Nathalie; Spotheim-Maurizot, Mélanie; Charlier, Michel

    2005-04-01

    The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects. PMID:15799700

  2. MicroRNA-145 Targets YES and STAT1 in Colon Cancer Cells

    PubMed Central

    Gregersen, Lea H.; Jacobsen, Anders B.; Frankel, Lisa B.; Wen, Jiayu; Krogh, Anders; Lund, Anders H.

    2010-01-01

    Background MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. Methodology/Principal Findings To investigate the role of miR-145 in colon cancer, we have employed a microarray based approach to identify miR-145 targets. Based on seed site enrichment analyses and unbiased word analyses, we found a significant enrichment of miRNA binding sites in the 3′-untranslated regions (UTRs) of transcripts down-regulated upon miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets. Conclusions/Significance The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor-suppressive functions of miR-145. PMID:20098684

  3. STAT1 regulates IFN-alpha beta- and IFN-gamma-dependent control of infection with Chlamydia pneumoniae by nonhemopoietic cells.

    PubMed

    Rothfuchs, Antonio Gigliotti; Trumstedt, Christian; Mattei, Fabrizio; Schiavoni, Giovanna; Hidmark, Asa; Wigzell, Hans; Rottenberg, Martín E

    2006-06-01

    STAT1 mediates signaling in response to IFN-alpha, -beta, and -gamma, cytokines required for protective immunity against several viral, bacterial, and eukaryotic pathogens. The protective role of STAT1 in the control of intranasal infection with the obligate intracellular bacterium Chlamydia pneumoniae was analyzed. IFN-gamma-/- or IFN-gamma receptor (R)-/- mice were highly susceptible to infection with C. pneumoniae. We found that STAT1-/- mice were even more susceptible to C. pneumoniae than IFN-gamma-/- or IFN-gammaR-/- mice. Phosphorylation of STAT1 was detected in the lungs of C. pneumoniae-infected wild-type, IFN-gammaR-/-, and IFN-alphabetaR-/- mice, but not in mice lacking both IFN-alphabetaR and IFN-gammaR. In line with this, IFN-alphabetaR-/-/IFN-gammaR-/- mice showed increased susceptibility to infection compared with IFN-gammaR-/- mice. However, C. pneumoniae-infected IFN-alphabetaR-/- or IFN regulatory factor 3-/- mice showed no increased susceptibility and similar IFN-gamma expression compared with wild-type mice. CD4+ or CD8+ cells released IFN-gamma in vivo and conferred protection against C. pneumoniae in a STAT1-independent manner. In contrast, STAT1 mediated a nonredundant protective role of nonhemopoietic cells but not of hemopoietic cells. Nonhemopoietic cells accounted for the expression of STAT1-mediated indoleamine 2, 3-dioxygenase and the p47 GTPase LRG-47, but not inducible NO synthase mRNA. In summary, we demonstrate that STAT1 mediates a cooperative effect of IFN-alphabeta and IFN-gamma on nonhemopoietic cells, resulting in protection against C. pneumoniae. PMID:16709859

  4. VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling

    PubMed Central

    Afroz, Sharmin; Brownlie, Robert; Fodje, Michel

    2016-01-01

    ABSTRACT The UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo. In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication

  5. IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche

    PubMed Central

    Pereira, Leticia; Medina, Rebeca; Baena, Miguel; Planas, Anna M.; Pozas, Esther

    2015-01-01

    The adult subventricular zone (SVZ) is the main neurogenic niche in normal adult brains of mice and rats. Interferon gamma (IFNγ) has somewhat controversially been associated with SVZ progenitor proliferation and neurogenesis. The in vivo involvement of IFNγ in the physiology of the adult SVZ niche is not fully understood and its intracellular mediators are unknown. Here we show that IFNγ, through activation of its canonical signal transducer and activator of transcription 1 (STAT1) pathway, acts specifically on Nestin+ progenitors by decreasing both progenitor proliferation and the number of cycling cells. In addition, IFNγ increases the number of neuroblasts generated without shifting glial fate determination. The final result is deficient recruitment of newborn neurons to the olfactory bulb (OB), indicating that IFNγ-induced stimulation of neuronal differentiation does not compensate for its antiproliferative effect. We conclude that IFNγ signaling via STAT1 in the SVZ acts dually as an antiproliferative and proneurogenic factor, and thereby regulates neurogenesis in normal adult brains. PMID:26217191

  6. Abnormal Mammary Development in 129:STAT1-Null Mice is Stroma-Dependent.

    PubMed

    Chen, Jane Q; Mori, Hidetoshi; Cardiff, Robert D; Trott, Josephine F; Hovey, Russell C; Hubbard, Neil E; Engelberg, Jesse A; Tepper, Clifford G; Willis, Brandon J; Khan, Imran H; Ravindran, Resmi K; Chan, Szeman R; Schreiber, Robert D; Borowsky, Alexander D

    2015-01-01

    Female 129:Stat1-null mice (129S6/SvEvTac-Stat1(tm1Rds) homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium. These abnormalities are partially reversed by elevated serum progesterone and prolactin whereas transplantation of wild-type bone marrow into 129:Stat1-null mice does not reverse the MG developmental defects. Medium conditioned by 129:Stat1-null epithelium-cleared MFP does not stimulate epithelial proliferation, whereas it is stimulated by medium conditioned by epithelium-cleared MFP from either wild-type or 129:Stat1-null females having elevated progesterone and prolactin. Microarrays and multiplexed cytokine assays reveal that the MG of 129:Stat1-null mice has lower levels of growth factors that have been implicated in normal MG growth and development. Transplanted 129:Stat1-null tumors and their isolated cells also grow slower in 129:Stat1-null MG compared to wild-type recipient MG. These studies demonstrate that growth of normal and neoplastic 129:Stat1-null epithelium is dependent on the hormonal milieu and on factors from the mammary stroma such as cytokines. While the individual or combined effects of these factors remains to be resolved, our data supports the role of STAT1 in maintaining a tumor-suppressive MG microenvironment.

  7. Molecular cloning and expression analysis of the STAT1 gene in the water buffalo (Bubalus bubalis).

    PubMed

    Deng, Tingxian; Pang, Chunying; Zhu, Peng; Liao, Biyun; Zhang, Ming; Yang, Bingzhuang; Liang, Xianwei

    2015-01-01

    Signal transducer and activator of transcription 1 (STAT1) is a critical component of the transcription factor complex in the interferon (IFN) signaling pathways. Of the seven STAT isoforms, STAT1 is a key mediator of type I and type III IFN signaling, but limited information is available for the STAT genes in the water buffalo. Here, we amplified and identified the complete coding sequence (CDS) of the buffalo STAT1 gene by using reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that the buffalo STAT1 gene length size was 3437 bp, containing an open reading frame (ORF) of 2244 bp that encoded 747 amino acids for the first time. The buffalo STAT1 CDS showed 99, 98, 89, 93, 86, 85, and 87% identity with that of Bos taurus, Ovis aries, Homo sapiens, Sus scrofa, Rattus norvegicus, Mus musculus, and Capra hircus. The phylogenetic analyses revealed that the nearest relationship existed between the water buffalo and B. taurus. The STAT1 gene was ubiquitously expressed in 11 buffalo tissues by real-time PCR, whereas STAT1 was expressed at higher levels in the lymph. The STAT1 gene contained five targeted microRNA sequences compared with the B. taurus by the miRBase software that provide a fundamental for identifying the STAT1 gene function.

  8. Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

    PubMed Central

    Wang, Shuo; Patsis, Christos; Koromilas, Antonis E.

    2015-01-01

    The signal transducer and activator of transcription 1 (Stat1) functions as a tumor suppressor via immune regulatory and cell-autonomous pathways. Herein, we report a previously unidentified cell-autonomous Stat1 function, which is its ability to exhibit both antiproliferative and prosurvival properties by facilitating translation of mRNAs encoding for the cyclin-dependent kinase inhibitor p27Kip1 and antiapoptotic proteins X-linked inhibitor of apoptosis and B-cell lymphoma xl. Translation of the select mRNAs requires the transcriptional function of Stat1, resulting in the up-regulation of the p110γ subunit of phosphoinositide 3-kinase (PI3K) class IB and increased expression of the translational repressor translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). Increased PI3Kγ signaling promotes the degradation of the eIF4A inhibitor programmed cell death protein 4, which favors the cap-independent translation of the select mRNAs under conditions of general inhibition of protein synthesis by up-regulated eIF4E-binding protein 1. As such, Stat1 inhibits cell proliferation but also renders cells increasingly resistant to antiproliferative effects of pharmacological inhibitors of PI3K and/or mammalian target of rapamycin. Stat1 also protects Ras-transformed cells from the genotoxic effects of doxorubicin in culture and immune-deficient mice. Our findings demonstrate an important role of mRNA translation in the cell-autonomous Stat1 functions, with implications in tumor growth and treatment with chemotherapeutic drugs. PMID:25870277

  9. Tau Induces Cooperative Taxol Binding to Microtubules

    NASA Astrophysics Data System (ADS)

    Ross, Jennifer; Santangelo, Christian; Victoria, Makrides; Fygenson, Deborah

    2004-03-01

    Taxol and tau are two ligands which stabilize the microtubule (MT) lattice. Taxol is an anti-mitotic drug that binds β tubulin in the MT interior. Tau is a MT-associated protein that binds both α and β tubulin on the MT exterior. Both taxol and tau reduce MT dynamics and promote tubulin polymerization. Tau alone also acts as a buttress to bundle, stiffen, and space MTs. A structural study recently suggested that taxol and tau may interact by binding to the same site. Using fluorescence recovery after photobleaching, we find that tau induces taxol to bind MTs cooperatively depending on the tau concentration. We develop a model that correctly fits the data in the absence of tau and yields a measure of taxol cooperativity when tau is present.

  10. Glucolipotoxicity initiates pancreatic β-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation.

    PubMed

    Bagnati, Marta; Ogunkolade, Babatunji W; Marshall, Catriona; Tucci, Carmen; Hanna, Katie; Jones, Tania A; Bugliani, Marco; Nedjai, Belinda; Caton, Paul W; Kieswich, Julius; Yaqoob, Muhammed M; Ball, Graham R; Marchetti, Piero; Hitman, Graham A; Turner, Mark D

    2016-01-01

    Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing β-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the β-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in β-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death. PMID:27512950

  11. The measles virus phosphoprotein interacts with the linker domain of STAT1

    SciTech Connect

    Devaux, Patricia Priniski, Lauren; Cattaneo, Roberto

    2013-09-15

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  12. Putting the brakes on mammary tumorigenesis: Loss of STAT1 predisposes to intraepithelial neoplasias

    PubMed Central

    Schneckenleithner, Christine; Bago-Horvath, Zsuzsanna; Dolznig, Helmut; Neugebauer, Nina; Kollmann, Karoline; Kolbe, Thomas; Decker, Thomas; Kerjaschki, Dontscho; Wagner, Kay-Uwe; Müller, Mathias; Stoiber, Dagmar; Sexl, Veronika

    2011-01-01

    Multiparous Stat1−/− mice spontaneously develop mammary tumors with increased incidence: at an average age of 12 months, 55% of the animals suffer from mammary cancer, although the histopathology is heterogeneous. We consistently observed mosaic expression or down-regulation of STAT1 protein in wild-type mammary cancer evolving in the control group. Transplantation experiments show that tumorigenesis in Stat1−/− mice is partially influenced by impaired CTL mediated tumor surveillance. Additionally, STAT1 exerts an intrinsic tumor suppressing role by controlling and blocking proliferation of the mammary epithelium. Loss of STAT1 in epithelial cells enhances cell growth in both transformed and primary cells. The increased proliferative capacity leads to the loss of structured acini formation in 3D-cultures. Analogous effects were observed when Irf1−/− epithelial cells were used. Accordingly, the rate of mammary intraepithelial neoplasias (MINs) is increased in Stat1−/− animals: MINs represent the first step towards mammary tumors. The experiments characterize STAT1/IRF1 as a key growth inhibitory and tumor suppressive signaling pathway that prevents mammary cancer formation by maintaining growth control. Furthermore, they define the loss of STAT1 as a predisposing event via enhanced MIN formation. PMID:22185785

  13. The non-pathogenic Henipavirus Cedar paramyxovirus phosphoprotein has a compromised ability to target STAT1 and STAT2.

    PubMed

    Lieu, Kim G; Marsh, Glenn A; Wang, Lin-Fa; Netter, Hans J

    2015-12-01

    Immune evasion by the lethal henipaviruses, Hendra (HeV) and Nipah virus, is mediated by its interferon (IFN) antagonist P gene products, phosphoprotein (P), and the related V and W proteins, which can target the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins to inhibit IFN/STAT signaling. However, it is not clear if the recently identified non-pathogenic Henipavirus, Cedar paramyxovirus (CedPV), is also able to antagonize the STAT proteins. We performed comparative studies between the HeV P gene products (P/V/W) and CedPV-P (CedPV does not encode V or W) and demonstrate that differences exist in their ability to engage the STAT proteins using immunoprecipitation and quantitative confocal microscopic analysis. In contrast to HeV-P gene encoded proteins, the ability of CedPV-P to interact with and relocalize STAT1 or STAT2 is compromised, correlating with a reduced capacity to inhibit the mRNA synthesis of IFN-inducible gene MxA. Furthermore, infection studies with HeV and CedPV demonstrate that HeV is more potent than CedPV in inhibiting the IFN-α-mediated nuclear accumulation of STAT1. These results strongly suggest that the ability of CedPV to counteract the IFN/STAT response is compromised compared to HeV.

  14. A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-γ-mediated responses in chronic mucocutaneous candidiasis.

    PubMed

    Martinez-Martinez, Laura; Martinez-Saavedra, Maria Teresa; Fuentes-Prior, Pablo; Barnadas, Maria; Rubiales, Maria Victoria; Noda, Judith; Badell, Isabel; Rodríguez-Gallego, Carlos; de la Calle-Martin, Oscar

    2015-12-01

    Gain-of-function STAT1 mutations have recently been associated with autosomal dominant chronic mucocutaneous candidiasis (CMC). The purpose of this study was to characterize the three members of a non-consanguineous family, the father and his two sons, who presented with recurrent oral thrush and ocular candidiasis since early childhood. The three patients had reduced levels of IL-17-producing T cells. This reduction affected specifically IL-17(+)IFN-γ(-) T cells, because the levels of IL-17(+)IFN-γ(+) T cells were similar to controls. We found that PBMC (peripheral blood mononuclear cells) from the patients did not respond to Candida albicans ex vivo. Moreover, after polyclonal activation, patients' PBMC produced lower levels of IL-17 and IL-6 and higher levels of IL-4 than healthy controls. Genetic analyses showed that the three patients were heterozygous for a new mutation in STAT1 (c.894A>C, p.K298N) that affects a highly conserved residue of the coiled-coil domain of STAT1. STAT1 phosphorylation levels were significantly higher in patients' cells than in healthy controls, both in basal conditions and after IFN-γ stimulation, suggesting a permanent activation of STAT1. Cells from the patients also presented increased IFN-γ-mediated responses measured as MIG and IP-10 production. In conclusion, we report a novel gain-of-function mutation in the coiled-coil domain of STAT1, which increases STAT1 phosphorylation and impairs IL-17-mediated immunity. The mutation is responsible for CMC in this family with autosomal dominant inheritance of the disease.

  15. Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

    PubMed Central

    Park, Eun-Mi; Kang, Jung-Ha; Seo, Jung Soo; Kim, GunDo; Chung, Jongkyeong; Choi, Tae-Jin

    2008-01-01

    Background Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. Results A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. Conclusion Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish. PMID:18578892

  16. Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis

    PubMed Central

    Mizoguchi, Yoko; Tsumura, Miyuki; Okada, Satoshi; Hirata, Osamu; Minegishi, Shizuko; Imai, Kohsuke; Hyakuna, Nobuyuki; Muramatsu, Hideki; Kojima, Seiji; Ozaki, Yusuke; Imai, Takehide; Takeda, Sachiyo; Okazaki, Tetsuya; Ito, Tsuyoshi; Yasunaga, Shin'ichiro; Takihara, Yoshihiro; Bryant, Vanessa L.; Kong, Xiao-Fei; Cypowyj, Sophie; Boisson-Dupuis, Stéphanie; Puel, Anne; Casanova, Jean-Laurent; Morio, Tomohiro; Kobayashi, Masao

    2014-01-01

    CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14+ cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations. PMID:24343863

  17. Tau binds ATP and induces its aggregation.

    PubMed

    Farid, Mina; Corbo, Christopher P; Alonso, Alejandra Del C

    2014-02-01

    Tau is a microtubule-associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well-defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self-assembly. We found that tau's N-terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self-assembly into filaments. At 1 mM ATP the filaments are 4-7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP-induced self-assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly. PMID:24258797

  18. Small-Molecule Inhibitors of Cytokine-Mediated STAT1 Signal Transduction In β-Cells With Improved Aqueous Solubility

    PubMed Central

    Scully, Stephen S.; Tang, Alicia J.; Lundh, Morten; Mosher, Carrie M.; Perkins, Kedar M.; Wagner, Bridget K.

    2013-01-01

    We previously reported the discovery of BRD0476 (1), a small molecule generated by diversity-oriented synthesis that suppresses cytokine-induced β-cell apoptosis. Herein, we report the synthesis and biological evaluation of 1 and analogs with improved aqueous solubility. By replacing naphthyl with quinoline moieties, we prepared active analogs with up to a 1400-fold increase in solubility from 1. In addition, we demonstrated that compound 1 and analogs inhibit STAT1 signal transduction induced by IFN-γ. PMID:23617753

  19. Triggering of Toll-like receptors modulates IFN-gamma signaling: involvement of serine 727 STAT1 phosphorylation and suppressors of cytokine signaling.

    PubMed

    Dalpke, Alexander H; Eckerle, Susan; Frey, Markus; Heeg, Klaus

    2003-07-01

    Microbial stimuli activate cells of the innate immune system by triggering Toll-like receptors (TLR). Activation of macrophages and dendritic cells is further enhanced by secondary signals like IFN-gamma. Here we analyzed the interplay of IFN-gamma and TLR signaling in cells of the innate immune system. Using a STAT1-dependent reporter construct we show that IFN-gamma signaling can be enhanced as well as inhibited by simultaneous stimulation with either defined TLR agonists or whole-bacterial lysates. Short costimulation resulted in the amplification of IFN-gamma signaling and was attributable to the p38 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of signal transducer and activator of transcription (STAT)1 on serine 727. In contrast, prolonged co-incubation as well as pre-incubation with TLR agonists led to an inhibition of IFN-gamma signaling. TLR triggering induced expression of suppressor of cytokine signaling (SOCS)-1, SOCS-3 and cytokine-inducible SH2 domain-containing protein (CIS). Overexpression of SOCS-1 and, to a lesser extend, of SOCS-3 and CIS inhibited IFN-gamma signaling as measured by activation of STAT1. Moreover, pre-incubation with TLR-dependent stimuli impaired IFN-gamma-induced MHC class II regulation but enhanced CD40 and CD86 expression. Taken together, the results indicate a tight interplay between TLR and IFN-gamma signaling pathways which involve induction of SOCS proteins and serine phosphorylation of STAT1.

  20. Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages.

    PubMed

    Cho, Byoung Ok; Yin, Hong Hua; Park, Sang Hyun; Byun, Eui Baek; Ha, Hun Yong; Jang, Seon Il

    2016-08-01

    Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE2, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages. PMID:27068250

  1. Active components with inhibitory activities on IFN-γ/STAT1 and IL-6/STAT3 signaling pathways from Caulis Trachelospermi.

    PubMed

    Liu, Xiao-Ting; Wang, Zhe-Xing; Yang, Yu; Wang, Lin; Sun, Ruo-Feng; Zhao, Yi-Min; Yu, Neng-Jiang

    2014-01-01

    Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1) with IC50 value of 2.43 μg/mL as well as inhibiting IL-6-induced STAT3-responsive luciferase activity (IL-6/STAT3) with IC50 value of 1.38 μg/mL. Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1) and nortrachelogenin 4-O-β-D-glucopyranoside (2), together with six known compounds. The lignan compounds 1-4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively. PMID:25100250

  2. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    PubMed Central

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  3. MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

    SciTech Connect

    Li, Jun; He, Xijing; Wei, Wenzhi; Zhou, Xiaobo

    2015-05-01

    Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a direct interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation. - Highlights: • Overexpression of miR-194 significantly increased osteoblast differentiation. • miR-194 directly targeted the 3′- UTR of STAT1. • miR-194 regulated the expression of STAT1. • Overexpression of miR-194 promoted the nuclear translocation of Runx2.

  4. STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis

    PubMed Central

    Kim, Minji; O'Brien, Lucy Erin; Kwon, Sang-Ho

    2010-01-01

    Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial–mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal. PMID:20861313

  5. Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts

    PubMed Central

    Sobhkhez, Mehrdad; Skjesol, Astrid; Thomassen, Ernst; Tollersrud, Linn Greiner; Iliev, Dimitar B.; Sun, Baojian; Robertsen, Børre; Jørgensen, Jorunn B.

    2014-01-01

    Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish. PMID:25379383

  6. Lactation-induced cadmium-binding proteins

    SciTech Connect

    Bhattacharyya, M.H.; Solaiman, D.; Garvey, J.S.; Miyazaki, W.Y.

    1987-01-01

    Previously we have demonstrated an increase during midlactation in /sup 109/Cd adsorption and increased retention by the duodenum, kidney, and mammary tissue of mouse dams receiving environmental levels of cadmium//sup 109/Cd via drinking water, with little change in /sup 109/Cd retention in liver and jejunum compared to nonpregnant controls. Results are reported here of a study of cadmium deposition during midlactation as associated with induction of metallothionein (MT). A cadmium/hemoglobin (Cd/Hb) assay and radioimmunoassay for MT which measures heat-stable cadmium binding capacity in tissues was used to determine MT concentrations in fractions of kidney, liver, duodenum, and jejunum from female mice. Both assays demonstrated clear lactation-induced increases in MT concentrations in liver, kidney, and duodenum, with MT concentrations falling rapidly to control levels after weaning. 4 refs., 1 tab.

  7. Fibroblast growth factor inhibits interferon γ-STAT1 and interleukin 6-STAT3 signaling in chondrocytes

    PubMed Central

    Krejci, Pavel; Prochazkova, Jirina; Bryja, Vitezslav; Jelinkova, Petra; Pejchalova, Katerina; Kozubik, Alois; Thompson, Leslie Michels; Wilcox, William R.

    2008-01-01

    Activation of fibroblast growth factor receptor 3 (FGFR3) leads to attenuation of cartilage growth. The members of the STAT family of transcription factors are believed to participate in FGFR3 signaling in cartilage, however the molecular mechanism of this action is poorly understood. Here, we demonstrate that a chronic FGF stimulus leads to accumulation of STAT1, 3, 5 and 6, evident in both in vitro chondrocyte model and murine limb explant cultures. Despite the accumulation, both endogenous and cytokine-induced activation of STAT1 and STAT3 is impaired by FGF, as demonstrated by imaging of active STAT nuclear translocation and analyses of STAT activatory phosphorylation and transcriptional activation. Further, we demonstrate that FGF induces expression of CIS, SOCS1 and SOCS3 inhibitors of gp130, a common receptor for the IL6-family of cytokines. Since cytokine-gp130 signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth-inhibitory effect of FGFR3 in cartilage. PMID:18950705

  8. Aqueous extract of Arbutus unedo inhibits STAT1 activation in human breast cancer cell line MDA-MB-231 and human fibroblasts through SHP2 activation.

    PubMed

    Mariotto, S; Ciampa, A R; de Prati, A Carcereri; Darra, E; Vincenzi, S; Sega, M; Cavalieri, E; Shoji, K; Suzuki, H

    2008-05-01

    Arbutus unedo L. has been for a long time employed in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes. Signal transducer and activator of transcription 1 (STAT1) is a fascinating and complex protein with multiple yet contrasting transcriptional functions. Although activation of this nuclear factor is finely regulated in order to control the entire inflammatory process, its hyper-activation or time-spatially erroneous activation may lead to exacerbation of inflammation. The modulation of this nuclear factor, therefore, has recently been considered as a new strategy in the treatment of inflammatory diseases. In this study, we present data showing that the aqueous extract of Arbutus unedo's leaves exerts inhibitory action on interferon-gamma (IFN-gamma) elicited activation of STAT1, both in human breast cancer cell line MDA-MB-231 and in human fibroblasts. This down-regulation of STAT1 is shown to result from a reduced tyrosine phosphorylation of STAT1 protein. Evidence is also presented indicating that the inhibitory effect of this extract may be mediated through enhancement of tyrosine phosphorylation of SHP2 tyrosine phosphatase. The modulation of this nuclear factor turns out into the regulation of the expression of a number of genes involved in the inflammatory response such as inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1). Taken together, our results suggest that the employment of the Arbutus unedo aqueous extract is promising, at least, as an auxiliary anti-inflammatory treatment of diseases in which STAT1 plays a critical role. PMID:18473914

  9. STAT1 and STAT3 in tumorigenesis: A matter of balance.

    PubMed

    Avalle, Lidia; Pensa, Sara; Regis, Gabriella; Novelli, Francesco; Poli, Valeria

    2012-04-01

    The transcription factors STAT1 and STAT3 appear to play opposite roles in tumorigenesis. While STAT3 promotes cell survival/proliferation, motility and immune tolerance and is considered as an oncogene, STAT1 mostly triggers anti-proliferative and pro-apoptotic responses while enhancing anti-tumor immunity. Despite being activated downstream of common cytokine and growth factor receptors, their activation is reciprocally regulated and perturbation in their balanced expression or phosphorylation levels may re-direct cytokine/growth factor signals from proliferative to apoptotic, or from inflammatory to anti-inflammatory. Here we review the functional canonical and non-canonical effects of STAT1 and STAT3 activation in tumorigenesis and their potential cross-regulation mechanisms.

  10. Signal transducer and activator of transcription 1 (STAT-1) plays a critical role in control of Trypanosoma cruzi infection.

    PubMed

    Kulkarni, Manjusha M; Varikuti, Sanjay; Terrazas, Cesar; Kimble, Jennifer L; Satoskar, Abhay R; McGwire, Bradford S

    2015-06-01

    The control of Trypanosoma cruzi infection is related to interferon-γ (IFN-γ) activation leading to intracellular clearance of parasites. The transcription factor signal transducer and activator of transcription 1 (STAT-1) is a key mediator of IFN-γ intracellular signalling and knockout of this protein leads to susceptibility to several intracellular microbes. To determine the role of STAT-1 in host susceptibility to T. cruzi infection we compared the survival, parasite loads and balance of IFN-γ and interleukin-10 (IL-10) responses between wild-type and STAT-1 knockout mice. We found that the lack of STAT-1 resulted in a more robust infection, leading to higher levels of blood and tissue parasites and markedly reduced survival. In addition, infected STAT-1 knockout mice had higher systemic levels of both IFN-γ and IL-10, suggesting that the absence of STAT-1 leads to a disequilibrium of pro-inflammatory and anti-inflammatory cytokines. Analysis of spleen cells indicates that CD4, CD8 cells generate IFN-γ and natural killer cells express IL-13 in STAT-1 knockout animals. The production of IL-17 is particularly enhanced in the absence STAT-1 expression but did not reduce mortality. Overall these results indicate that STAT-1 is important for the control of T. cruzi infection in mice.

  11. Transcriptional regulation of oncogenic protein kinase Cϵ (PKCϵ) by STAT1 and Sp1 proteins.

    PubMed

    Wang, HongBin; Gutierrez-Uzquiza, Alvaro; Garg, Rachana; Barrio-Real, Laura; Abera, Mahlet B; Lopez-Haber, Cynthia; Rosemblit, Cinthia; Lu, Huaisheng; Abba, Martin; Kazanietz, Marcelo G

    2014-07-11

    Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼ 1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and -105 bp (region A) and -921 and -796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and -269/-247 as well as STAT1 sites in positions -880/-869 and -793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.

  12. Signal transducer and activator of transcription 1 (STAT1) acts like an oncogene in malignant pleural mesothelioma.

    PubMed

    Arzt, Lisa; Kothmaier, Hannelore; Halbwedl, Iris; Quehenberger, Franz; Popper, Helmut H

    2014-07-01

    Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is increasing in Europe and the prognosis remains poor. We compared epithelioid MPM in short and long survivors, and identified signal transducer and activator of transcription 1 (STAT1) as probably being responsible for antiapoptotic signaling and chemoresistance. Six mesothelioma cell lines were evaluated by Western Blot. We also analyzed 16 epithelioid MPM tissue samples for the phosphorylation status of STAT1 and the expression of its negative regulator, the suppressor of cytokine signaling 1 (SOCS1). Formalin-fixed and paraffin-embedded tissue specimens were evaluated by protein-lysate microarray and immunohistochemistry. We found STAT1 to be highly expressed and STAT3 downregulated in MPM cell lines. The expression of STAT1 phosphorylated on tyrosine 701 (Y701) was increased by interferon-gamma (IFN-γ) treatment, whereas SOCS1 was not expressed. The expression of STAT1 phosphorylated on serine 727 (S727) was not detected in mesothelioma cell lines and was not stimulated by IFN-γ. STAT1 was phosphorylated on tyrosine 701 and serine 727 in MPM tissue samples. The expression of pSTAT1-Y701 was increased compared to pSTAT1-S727. SOCS1 was again not detectable. STAT1 is upregulated in MPM, and its action may be prolonged by a loss of the negative regulator SOCS1. STAT1 might, therefore, be a target for therapeutic intervention, with the intention to restore apoptotic mechanisms and sensitivity to chemotherapy. However, other regulatory mechanisms need to be investigated to clarify if lack of expression of SOCS1 is the only reason for sustained STAT1 expression in MPM.

  13. Fucose-binding Lotus tetragonolobus lectin binds to human polymorphonuclear leukocytes and induces a chemotactic response.

    PubMed

    VanEpps, D E; Tung, K S

    1977-09-01

    Fucose-binding L. tetragonolobus lectin to the surface of human polymorphonuclear leukocytes (PMN) and induces a chemotactic response. Both surface binding and chemotaxis are inhibited by free fucose but not by fructose, mannose, or galactose. The lectin-binding sites on PMN are unrelated to the A, B, or O blood group antigen. Utilization of this lectin should be a useful tool in isolating PMN membrane components and in analyzing the mechanism of neutrophil chemotaxis. PMID:330752

  14. Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors

    PubMed Central

    Schaal, Courtney; Chellappan, Srikumar

    2016-01-01

    Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine–mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine. PMID:27228072

  15. Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors.

    PubMed

    Schaal, Courtney; Chellappan, Srikumar

    2016-01-01

    Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine-mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine. PMID:27228072

  16. The Ets-1 transcription factor is required for Stat1-mediated T-bet expression and IgG2a class switching in mouse B cells.

    PubMed

    Nguyen, Hai Vu; Mouly, Enguerran; Chemin, Karine; Luinaud, Romain; Despres, Raymonde; Fermand, Jean-Paul; Arnulf, Bertrand; Bories, Jean-Christophe

    2012-05-01

    In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell-independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1-deficient (Ets-1(-/-)) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1(-/-) B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells.

  17. Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1.

    PubMed

    Hogan, Robert J; Gao, Guangping; Rowe, Thomas; Bell, Peter; Flieder, Douglas; Paragas, Jason; Kobinger, Gary P; Wivel, Nelson A; Crystal, Ronald G; Boyer, Julie; Feldmann, Heinz; Voss, Thomas G; Wilson, James M

    2004-10-01

    Intranasal inhalation of the severe acute respiratory syndrome coronavirus (SARS CoV) in the immunocompetent mouse strain 129SvEv resulted in infection of conducting airway epithelial cells followed by rapid clearance of virus from the lungs and the development of self-limited bronchiolitis. Animals resistant to the effects of interferons by virtue of a deficiency in Stat1 demonstrated a markedly different course following intranasal inhalation of SARS CoV, one characterized by replication of virus in lungs and progressively worsening pulmonary disease with inflammation of small airways and alveoli and systemic spread of the virus to livers and spleens.

  18. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes

    PubMed Central

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161− subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  19. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes.

    PubMed

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161- subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  20. Differential methylation hybridization profiling identifies involvement of STAT1-mediated pathways in breast cancer.

    PubMed

    Kim, Ju Hee; Kang, Han-Sung; Kim, Tae Woo; Kim, Sun Jung

    2011-10-01

    Many cancer-related genes are regulated by an epigenetic mechanism through modification of the methylation status of CpG sites at the promoter. This study was carried out at a genome-wide scale to mine genes in which the methylation of CpG sites is altered in breast cancer tissues. Differential methylation hybridization analysis was conducted using a chromosomal DNA mixture of ten normal and cancer tissue sets. A CpG microarray harboring 237,220 CpG sites of the whole genome was interrogated and the resulting methylation level differences, as well as the RNA expression differences, between the normal and cancer sets for selected genes were verified in breast cell lines by methylation-specific PCR and real-time PCR analyses. As a result, we identified and verified novel genes that were hypermethylated in breast cancer, such as NRN1, CA5B and RPIA. Pathway analysis of the genes with altered methylation patterns identified the involvement of a differentiation-related network of genes whose activity may be heavily regulated by STAT1 in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to STAT1-dependent cellular differentiation may be intimately involved in breast carcinogenesis. These findings lend credence to the possibility of using tumor-specific alterations in methylation patterns as biomarkers in estimating prognosis and assessing treatment options for breast cancer. PMID:21674123

  1. Skin microbiome imbalance in patients with STAT1/STAT3 defects impairs innate host defense responses

    PubMed Central

    Smeekens, Sanne P.; Huttenhower, Curtis; Riza, Anca; van de Veerdonk, Frank; Zeeuwen, Patrick L.J.M.; Schalkwijk, Joost; van der Meer, Jos W.M.; Xavier, Ramnik J.; Netea, Mihai G.; Gevers, Dirk

    2014-01-01

    Background Chronic mucocutaneous candidiasis (CMC) and hyper IgE syndrome (HIES) are primary immunodeficiencies mainly caused by mutations in STAT1 and STAT3, respectively. CMC and HIES patients have an increased risk for skin and mucosal infections with fungal pathogens and Staphylococcus aureus. However, it is unknown whether the genetic defects in these patients also affect the skin and mucosal microbiome, which in turn may influence host defense mechanisms. Methods The skin and oral microbiome of CMC and HIES patients was compared to that of healthy controls at five body sites using 16S rRNA sequencing. The influence of skin colonizers on the immune response was investigated using in-vitro experiments. Results The microbiome of CMC and HIES patients contained more Gram-negative bacteria, especially Acinetobacter spp, and less of the normal Corynebacterium spp., compared to healthy controls. Exposure of human primary leukocytes to Acinetobacter suppressed the cytokine response to C. albicans and S. aureus, while the normal Corynebacteria did not suppress cytokine responses. Discussion These results demonstrate that central mediators of immune responses like STAT1 and STAT3 not only directly influence immune responses, but also result in changes of the skin microbiome that in turn can amplify the defective immune response against fungal and microbial pathogens. PMID:23796786

  2. Role of methyl-induced polarization in ion binding

    PubMed Central

    Rossi, Mariana; Tkatchenko, Alexandre; Rempe, Susan B.; Varma, Sameer

    2013-01-01

    The chemical property of methyl groups that renders them indispensable to biomolecules is their hydrophobicity. Quantum mechanical studies undertaken here to understand the effect of point substitutions on potassium (K-) channels illustrate quantitatively how methyl-induced polarization also contributes to biomolecular function. K- channels regulate transmembrane salt concentration gradients by transporting K+ ions selectively. One of the K+ binding sites in the channel’s selectivity filter, the S4 site, also binds Ba2+ ions, which blocks K+ transport. This inhibitory property of Ba2+ ions has been vital in understanding K-channel mechanism. In most K-channels, the S4 site is composed of four threonine amino acids. The K channels that carry serine instead of threonine are significantly less susceptible to Ba2+ block and have reduced stabilities. We find that these differences can be explained by the lower polarizability of serine compared with threonine, because serine carries one less branched methyl group than threonine. A T→S substitution in the S4 site reduces its polarizability, which, in turn, reduces ion binding by several kilocalories per mole. Although the loss in binding affinity is high for Ba2+, the loss in K+ binding affinity is also significant thermodynamically, which reduces channel stability. These results highlight, in general, how biomolecular function can rely on the polarization induced by methyl groups, especially those that are proximal to charged moieties, including ions, titratable amino acids, sulfates, phosphates, and nucleotides. PMID:23878238

  3. Sakuranetin Inhibits Inflammatory Enzyme, Cytokine, and Costimulatory Molecule Expression in Macrophages through Modulation of JNK, p38, and STAT1

    PubMed Central

    Kim, Ki-Young

    2016-01-01

    Sakuranetin is flavonoid phytoalexin that serves as a plant antibiotic and exists in Prunus and several other plant species. Recently, we identified the anti-inflammatory effect of Prunus yedoensis and found that there were few studies on the potential anti-inflammatory activity of sakuranetin, one of the main constituents of Prunus yedoensis. Here, we isolated peritoneal macrophages from thioglycollate-injected mice and examined whether sakuranetin affected the response of the macrophages in response to lipopolysaccharide (LPS) plus interferon- (IFN-) γ or LPS only. Sakuranetin suppressed the synthesis of iNOS and COX2 in LPS/IFN-γ stimulated cells and the secretion of TNF-α, IL-6, and IL-12 in LPS stimulated cells. The surface expression of the costimulatory molecules, CD86 and CD40, was also decreased. Among the LPS-induced signaling molecules, STAT1, JNK, and p38 phosphorylation was attenuated. These findings are evidence that sakuranetin acts as anti-inflammatory flavonoid and further study is required to evaluate its in vivo efficacy. PMID:27668006

  4. Sakuranetin Inhibits Inflammatory Enzyme, Cytokine, and Costimulatory Molecule Expression in Macrophages through Modulation of JNK, p38, and STAT1.

    PubMed

    Kim, Ki-Young; Kang, Hee

    2016-01-01

    Sakuranetin is flavonoid phytoalexin that serves as a plant antibiotic and exists in Prunus and several other plant species. Recently, we identified the anti-inflammatory effect of Prunus yedoensis and found that there were few studies on the potential anti-inflammatory activity of sakuranetin, one of the main constituents of Prunus yedoensis. Here, we isolated peritoneal macrophages from thioglycollate-injected mice and examined whether sakuranetin affected the response of the macrophages in response to lipopolysaccharide (LPS) plus interferon- (IFN-) γ or LPS only. Sakuranetin suppressed the synthesis of iNOS and COX2 in LPS/IFN-γ stimulated cells and the secretion of TNF-α, IL-6, and IL-12 in LPS stimulated cells. The surface expression of the costimulatory molecules, CD86 and CD40, was also decreased. Among the LPS-induced signaling molecules, STAT1, JNK, and p38 phosphorylation was attenuated. These findings are evidence that sakuranetin acts as anti-inflammatory flavonoid and further study is required to evaluate its in vivo efficacy. PMID:27668006

  5. Sakuranetin Inhibits Inflammatory Enzyme, Cytokine, and Costimulatory Molecule Expression in Macrophages through Modulation of JNK, p38, and STAT1

    PubMed Central

    Kim, Ki-Young

    2016-01-01

    Sakuranetin is flavonoid phytoalexin that serves as a plant antibiotic and exists in Prunus and several other plant species. Recently, we identified the anti-inflammatory effect of Prunus yedoensis and found that there were few studies on the potential anti-inflammatory activity of sakuranetin, one of the main constituents of Prunus yedoensis. Here, we isolated peritoneal macrophages from thioglycollate-injected mice and examined whether sakuranetin affected the response of the macrophages in response to lipopolysaccharide (LPS) plus interferon- (IFN-) γ or LPS only. Sakuranetin suppressed the synthesis of iNOS and COX2 in LPS/IFN-γ stimulated cells and the secretion of TNF-α, IL-6, and IL-12 in LPS stimulated cells. The surface expression of the costimulatory molecules, CD86 and CD40, was also decreased. Among the LPS-induced signaling molecules, STAT1, JNK, and p38 phosphorylation was attenuated. These findings are evidence that sakuranetin acts as anti-inflammatory flavonoid and further study is required to evaluate its in vivo efficacy.

  6. Cell-intrinsic role for IFN-α–STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

    PubMed Central

    Li, Haiyan S.; Gelbard, Alexander; Martinez, Gustavo J.; Esashi, Eiji; Zhang, Huiyuan; Nguyen-Jackson, Hoainam; Liu, Yong-Jun; Overwijk, Willem W.

    2011-01-01

    Plasmacytoid dendritic cells (pDCs) reside in bone marrrow and lymphoid organs in homeostatic conditions and typically secrete abundant quantities of type I interferons (IFNs) on Toll-like receptor triggering. Recently, a pDC population was identified within Peyer patches (PPs) of the gut that is distinguished by its lack of IFN production; however, the relationship of PP pDCs to pDCs in other organs has been unclear. We report that PP pDCs are derived from common DC progenitors and accumulate in response to Fms-like tyrosine kinase 3 ligand, yet appear divergent in transcription factor profile and surface marker phenotype, including reduced E2-2 and CCR9 expression. Type I IFN signaling via STAT1 has a cell-autonomous role in accrual of PP pDCs in vivo. Moreover, IFN-α enhances pDC generation from DC progenitors by a STAT1-dependent mechanism. pDCs that have been developed in the presence of IFN-α resemble PP pDCs, produce inflammatory cytokines, stimulate Th17 cell generation, and fail to secrete IFN-α on Toll-like receptor engagement. These results indicate that IFN-α influences the development and function of pDCs by inducing emergence of an inflammatory (Th17-inducing) antigen-presenting subset, and simultaneously regulating accumulation of pDCs in the intestinal microenvironment. PMID:21828128

  7. Loss of STAT1 protects hair cells from ototoxicity through modulation of STAT3, c-Jun, Akt, and autophagy factors.

    PubMed

    Levano, S; Bodmer, D

    2015-01-01

    Hair cell damage is a side effect of cisplatin and aminoglycoside use. The inhibition or attenuation of this process is a target of many investigations. There is growing evidence that STAT1 deficiency decreases cisplatin-mediated ototoxicity; however, the role of STAT function and the molecules that act in gentamicin-mediated toxicity have not been fully elucidated. We used mice lacking STAT1 to investigate the effect of STAT1 ablation in cultured organs treated with cisplatin and gentamicin. Here we show that ablation of STAT1 decreased cisplatin toxicity and attenuated gentamicin-mediated hair cell damage. More TUNEL-positive hair cells were observed in explants of wild-type mice than that of STAT1(-/-) mice. Although cisplatin increased serine phosphorylation of STAT1 in wild-type mice and diminished STAT3 expression in wild-type and STAT1(-/-) mice, gentamicin increased tyrosine phosphorylation of STAT3 in STAT1(-/-) mice. The early inflammatory response was manifested in the upregulation of TNF-α and IL-6 in cisplatin-treated explants of wild-type and STAT1(-/-) mice. Expression of the anti-inflammatory cytokine IL-10 was altered in cisplatin-treated explants, upregulated in wild-type explants, and downregulated in STAT1(-/-) explants. Cisplatin and gentamicin triggered the activation of c-Jun. Activation of Akt was observed in gentamicin-treated explants from STAT1(-/-) mice. Increased levels of the autophagy proteins Beclin-1 and LC3-II were observed in STAT1(-/-) explants. These data suggest that STAT1 is a central player in mediating ototoxicity. Gentamicin and cisplatin activate different downstream factors to trigger ototoxicity. Although cisplatin and gentamicin triggered inflammation and activated apoptotic factors, the absence of STAT1 allowed the cells to overcome the effects of these drugs.

  8. Calreticulin binds to gentamicin and reduces drug-induced ototoxicity.

    PubMed

    Karasawa, Takatoshi; Wang, Qi; David, Larry L; Steyger, Peter S

    2011-12-01

    Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity. PMID:21785162

  9. Interferon-stimulated gene (ISG) 60, as well as ISG56 and ISG54, positively regulates TLR3/IFN-β/STAT1 axis in U373MG human astrocytoma cells.

    PubMed

    Imaizumi, Tadaatsu; Yoshida, Hidemi; Hayakari, Ryo; Xing, Fei; Wang, Lian; Matsumiya, Tomoh; Tanji, Kunikazu; Kawaguchi, Shogo; Murakami, Manabu; Tanaka, Hiroshi

    2016-04-01

    Treatment of cells with interferons (IFNs) induces the phosphorylation of signal transducer and activator of transcription 1 (STAT1), leading to the expression of hundreds of IFN-stimulated genes (ISGs). ISGs exert various antiviral and pro-inflammatory reactions. We have previously reported that ISG56 and ISG54 are induced by polyinosinic-polycytidylic acid (poly IC), an authentic agonist for Toll-like receptor 3 (TLR3), in U373MG human astrocytoma cells. ISG56 and ISG54 are also named as IFN-induced proteins with tetratricopeptide repeats (IFIT) 1 and IFIT2, respectively. In the present study, we demonstrated that poly IC induces the expression of ISG60, also named as IFIT3, in U373MG cells. RNA interference experiments showed that the induction of ISG60 by poly IC was mediated by TLR3, IFN-β, ISG56 and ISG54, whereas ISG60 is involved in poly IC-induced expression of ISG56, ISG54 and a chemokine CXCL10. The level of phosphorylated STAT1 was enhanced by poly IC, and it was inhibited by knockdown of ISG56, ISG54 or ISG60. These results suggest that there is a positive feedback loop between phosphorylated STAT1 and these ISGs. PMID:26423178

  10. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

    SciTech Connect

    Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto . E-mail: cattaneo.roberto@mayo.edu

    2007-03-30

    The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.

  11. Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.

    PubMed

    Toubiana, Julie; Okada, Satoshi; Hiller, Julia; Oleastro, Matias; Lagos Gomez, Macarena; Aldave Becerra, Juan Carlos; Ouachée-Chardin, Marie; Fouyssac, Fanny; Girisha, Katta Mohan; Etzioni, Amos; Van Montfrans, Joris; Camcioglu, Yildiz; Kerns, Leigh Ann; Belohradsky, Bernd; Blanche, Stéphane; Bousfiha, Aziz; Rodriguez-Gallego, Carlos; Meyts, Isabelle; Kisand, Kai; Reichenbach, Janine; Renner, Ellen D; Rosenzweig, Sergio; Grimbacher, Bodo; van de Veerdonk, Frank L; Traidl-Hoffmann, Claudia; Picard, Capucine; Marodi, Laszlo; Morio, Tomohiro; Kobayashi, Masao; Lilic, Desa; Milner, Joshua D; Holland, Steven; Casanova, Jean-Laurent; Puel, Anne

    2016-06-23

    Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A-producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis. PMID:27114460

  12. Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype

    PubMed Central

    Toubiana, Julie; Okada, Satoshi; Hiller, Julia; Oleastro, Matias; Lagos Gomez, Macarena; Aldave Becerra, Juan Carlos; Ouachée-Chardin, Marie; Fouyssac, Fanny; Girisha, Katta Mohan; Etzioni, Amos; Van Montfrans, Joris; Camcioglu, Yildiz; Kerns, Leigh Ann; Belohradsky, Bernd; Blanche, Stéphane; Bousfiha, Aziz; Rodriguez-Gallego, Carlos; Meyts, Isabelle; Kisand, Kai; Reichenbach, Janine; Renner, Ellen D.; Rosenzweig, Sergio; Grimbacher, Bodo; van de Veerdonk, Frank L.; Traidl-Hoffmann, Claudia; Picard, Capucine; Marodi, Laszlo; Morio, Tomohiro; Kobayashi, Masao; Lilic, Desa; Milner, Joshua D.; Holland, Steven; Casanova, Jean-Laurent

    2016-01-01

    Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A–producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis. PMID:27114460

  13. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells.

    PubMed

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l-/- MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  14. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

    PubMed Central

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l−/− MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  15. Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity

    PubMed Central

    Kint, Joeri; Dickhout, Annemiek; Kutter, Jasmin; Maier, Helena J.; Britton, Paul; Koumans, Joseph; Pijlman, Gorben P.; Fros, Jelke J.; Wiegertjes, Geert F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response. IMPORTANCE In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system. PMID:26401035

  16. Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

    PubMed Central

    Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

    2003-01-01

    The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

  17. Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-{alpha}/{beta} signaling

    SciTech Connect

    Caignard, Gregory; Guerbois, Mathilde; Labernardiere, Jean-Louis; Jacob, Yves; Jones, Louis M.; Wild, Fabian; Tangy, Frederic Vidalain, Pierre-Olivier

    2007-11-25

    Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-{alpha}/{beta}). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-{alpha}/{beta} pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-{alpha}/{beta} signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-{alpha}/{beta} receptor complex to block downstream signaling.

  18. Lectin KM+-induced neutrophil haptotaxis involves binding to laminin.

    PubMed

    Ganiko, Luciane; Martins, Antônio R; Freymüller, Edna; Mortara, Renato A; Roque-Barreira, Maria-Cristina

    2005-01-18

    The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both the extracellular matrix (ECM) and neutrophils depend on the lectin ability to recognize mannose-containing glycans. Here, we report the binding of KM+ to laminin and demonstrate that this interaction potentiates the KM+-induced neutrophil migration. Labeling of lung tissue by KM+ located its ligands on the endothelial cells, in the basement membrane, in the alveolus, and in the interstitial connective tissue. Such labeling was inhibited by 400 mM D-mannose, 10 mM Manalpha1-3[Manalpha1-6]Man or 10 microM peroxidase (a glycoprotein-containing mannosyl heptasaccharide). Laminin is a tissue ligand for KM+, since both KM+ and anti-laminin antibodies not only reacted with the same high molecular mass components of a lung extract, but also determined colocalized labeling in basement membranes of the lung tissue. The relevance of the KM+-laminin interaction to the KM+ property of inducing neutrophil migration was evaluated. The inability of low concentrations of soluble KM+ to induce human neutrophil migration was reversed by coating the microchamber filter with laminin. So, the interaction of KM+ with laminin promotes the formation of a substrate-bound KM+ gradient that is able to induce neutrophil haptotaxis.

  19. Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-γ Stimulated HaCaT Human Keratinocytes

    PubMed Central

    Kang, Na-Jin; Koo, Dong-Hwan; Kang, Gyeoung-Jin; Han, Sang-Chul; Lee, Bang-Won; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2015-01-01

    Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation. PMID:25995822

  20. Isorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation In Vivo and Human Bladder Cancer Invasion In Vitro by Targeting STAT1/FOXO1 Axis.

    PubMed

    Jiang, Guosong; Wu, Amy D; Huang, Chao; Gu, Jiayan; Zhang, Liping; Huang, Haishan; Liao, Xin; Li, Jingxia; Zhang, Dongyun; Zeng, Xingruo; Jin, Honglei; Huang, Haojie; Huang, Chuanshu

    2016-07-01

    Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1-FOXO1-MMP-2 axis. Cancer Prev Res; 9(7); 567-80. ©2016 AACR. PMID:27080594

  1. Superlattices assembled through shape-induced directional binding.

    PubMed

    Lu, Fang; Yager, Kevin G; Zhang, Yugang; Xin, Huolin; Gang, Oleg

    2015-04-23

    Organization of spherical particles into lattices is typically driven by packing considerations. Although the addition of directional binding can significantly broaden structural diversity, nanoscale implementation remains challenging. Here we investigate the assembly of clusters and lattices in which anisotropic polyhedral blocks coordinate isotropic spherical nanoparticles via shape-induced directional interactions facilitated by DNA recognition. We show that these polyhedral blocks--cubes and octahedrons--when mixed with spheres, promote the assembly of clusters with architecture determined by polyhedron symmetry. Moreover, three-dimensional binary superlattices are formed when DNA shells accommodate the shape disparity between nanoparticle interfaces. The crystallographic symmetry of assembled lattices is determined by the spatial symmetry of the block's facets, while structural order depends on DNA-tuned interactions and particle size ratio. The presented lattice assembly strategy, exploiting shape for defining the global structure and DNA-mediation locally, opens novel possibilities for by-design fabrication of binary lattices.

  2. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  3. Superlattices assembled through shape-induced directional binding

    SciTech Connect

    Lu, Fang; Yager, Kevin G.; Zhang, Yugang; Xin, Huolin; Gang, Oleg

    2015-04-23

    Organization of spherical particles into lattices is typically driven by packing considerations. Although the addition of directional binding can significantly broaden structural diversity, nanoscale implementation remains challenging. Here we investigate the assembly of clusters and lattices in which anisotropic polyhedral blocks coordinate isotropic spherical nanoparticles via shape-induced directional interactions facilitated by DNA recognition. We show that these polyhedral blocks—cubes and octahedrons—when mixed with spheres, promote the assembly of clusters with architecture determined by polyhedron symmetry. Moreover, three-dimensional binary superlattices are formed when DNA shells accommodate the shape disparity between nanoparticle interfaces. The crystallographic symmetry of assembled lattices is determined by the spatial symmetry of the block’s facets, while structural order depends on DNA-tuned interactions and particle size ratio. Lastly, the presented lattice assembly strategy, exploiting shape for defining the global structure and DNA-mediation locally, opens novel possibilities for by-design fabrication of binary lattices.

  4. Superlattices assembled through shape-induced directional binding

    PubMed Central

    Lu, Fang; Yager, Kevin G.; Zhang, Yugang; Xin, Huolin; Gang, Oleg

    2015-01-01

    Organization of spherical particles into lattices is typically driven by packing considerations. Although the addition of directional binding can significantly broaden structural diversity, nanoscale implementation remains challenging. Here we investigate the assembly of clusters and lattices in which anisotropic polyhedral blocks coordinate isotropic spherical nanoparticles via shape-induced directional interactions facilitated by DNA recognition. We show that these polyhedral blocks—cubes and octahedrons—when mixed with spheres, promote the assembly of clusters with architecture determined by polyhedron symmetry. Moreover, three-dimensional binary superlattices are formed when DNA shells accommodate the shape disparity between nanoparticle interfaces. The crystallographic symmetry of assembled lattices is determined by the spatial symmetry of the block's facets, while structural order depends on DNA-tuned interactions and particle size ratio. The presented lattice assembly strategy, exploiting shape for defining the global structure and DNA-mediation locally, opens novel possibilities for by-design fabrication of binary lattices. PMID:25903309

  5. Comparative proteomic analyses demonstrate enhanced interferon and STAT-1 activation in reovirus T3D-infected HeLa cells

    PubMed Central

    Ezzati, Peyman; Komher, Krysten; Severini, Giulia; Coombs, Kevin M.

    2015-01-01

    As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses. PMID:25905045

  6. STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

    PubMed Central

    Chmielewski, Stefan; Olejnik, Adam; Sikorski, Krzysztof; Pelisek, Jaroslav; Błaszczyk, Katarzyna; Aoqui, Cristiane; Nowicka, Hanna; Zernecke, Alma; Heemann, Uwe; Wesoly, Joanna; Baumann, Marcus; Bluyssen, Hans A. R.

    2014-01-01

    Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis. PMID:25478796

  7. Superlattices assembled through shape-induced directional binding

    DOE PAGES

    Lu, Fang; Yager, Kevin G.; Zhang, Yugang; Xin, Huolin; Gang, Oleg

    2015-04-23

    Organization of spherical particles into lattices is typically driven by packing considerations. Although the addition of directional binding can significantly broaden structural diversity, nanoscale implementation remains challenging. Here we investigate the assembly of clusters and lattices in which anisotropic polyhedral blocks coordinate isotropic spherical nanoparticles via shape-induced directional interactions facilitated by DNA recognition. We show that these polyhedral blocks—cubes and octahedrons—when mixed with spheres, promote the assembly of clusters with architecture determined by polyhedron symmetry. Moreover, three-dimensional binary superlattices are formed when DNA shells accommodate the shape disparity between nanoparticle interfaces. The crystallographic symmetry of assembled lattices is determined bymore » the spatial symmetry of the block’s facets, while structural order depends on DNA-tuned interactions and particle size ratio. Lastly, the presented lattice assembly strategy, exploiting shape for defining the global structure and DNA-mediation locally, opens novel possibilities for by-design fabrication of binary lattices.« less

  8. Inducible Cadmium Binding Complexes of Cabbage and Tobacco 1

    PubMed Central

    Wagner, George J.; Trotter, Melvin M.

    1982-01-01

    Cadmium complexes with apparent molecular weights of 10,000 were observed in aqueous extracts of Cd-treated cabbage (Brassica capitata L., cv. red danish) and tobacco (hybrid of Nicotiana glauca and N. langsdorffii) plants. The amount of complex (as Cd) recovered was found to be dependent on the concentration of the metal in the growth medium and the total time of exposure of plants to the metal. Induction of the complex at moderate levels of 112Cd exposure was monitored after labeling the complex with 109Cd in vitro. The constitutive nature of the ligand of the complex in cabbage and tobacco leaves was suggested when control plant extracts were exposed to 109Cd. Such extracts contained 109Cd, which eluted from Sephadex G-50 in the region of Cd complex. Simultaneous labeling with 112Cd and 35S or 32P indicated that the complex contained sulfur but probably not phosphorus. The amount of 35S which eluted coincident with 112Cd complex increased during complex induction. No evidence was found for the presence of 10,000 molecular weight Cd complex in stem exudates (vascular sap) of Cd-treated plants. The results obtained are consistent with the presence in these tissues of a ligand which is both inducible and constitutive and binds Cd in mercaptide bonds. All of these properties, and others reported earlier, are characteristic of Cd-metallothionein formed in animals. PMID:16662300

  9. Inducible cadmium binding complexes of cabbage and tobacco

    SciTech Connect

    Wagner, G.J.; Trotter, M.M.

    1982-01-01

    Cadmium complexes with apparent molecular weights of 10,000 were observed in aqueous extracts of Cd-treated cabbage (Brassica capitata L., cv. red danish) and tobacco (hybrid of Nicotiana glauca and N. langsdorffii) plants. The amount of complex (as Cd) recovered was found to be dependent on the concentration of the metal in the growth medium and the total time of exposure of plants to the metal. Induction of the complex at moderate levels of /sup 112/Cd exposure was monitored after labeling the complex with /sup 109/Cd in vitro. The constitutive nature of the ligand of the complex in cabbage and tobacco leaves was suggested when control plant extracts were exposed to /sup 109/Cd. Such extracts contained /sup 109/Cd, which eluted froom Sephadex G-50 in the region of Cd complex. Simultaneous labeling with /sup 112/Cd and /sup 35/S or /sup 32/P indicated that the complex contained sulfur but probably not phosphorus. The amount of /sup 35/S which eluted coincident with /sup 112/Cd complex increased during complex induction. No evidence was found for the presence of 10,000 molecular weight Cd complex in stem exudates (vascular sap) of Cd-treated plants. The results obtained are consistent with the presence in these tissues of a ligand which is both inducible and consitutive and binds Cd in mercaptide bonds. All of these properties and oters reported earlier, are characteristic of Cd-metallothionein formed in animals.

  10. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  11. BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation

    PubMed Central

    Iida, Ryuji; Bagavant, Harini; Alarcón-Riquelme, Marta E.

    2016-01-01

    The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease. PMID:27228057

  12. Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt's lymphoma Raji cells.

    PubMed

    Hosono, Masahiro; Sugawara, Shigeki; Matsuda, Atsushi; Tatsuta, Takeo; Koide, Yasuhiro; Hasan, Imtiaj; Hasan, Imtiaji; Ozeki, Yasuhiro; Nitta, Kazuo

    2014-10-01

    Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway. PMID:24861899

  13. Traditional herbal formula Jakyakgamcho-tang (Paeonia lactiflora and Glycyrrhiza uralensis) impairs inflammatory chemokine production by inhibiting activation of STAT1 and NF-κB in HaCaT cells.

    PubMed

    Jeong, Soo-Jin; Lim, Hye-Sun; Seo, Chang-Seob; Kim, Jung-Hoon; Jin, Seong-Eun; Yoo, Sae-Rom; Shin, Hyeun-Kyoo

    2015-02-15

    A traditional herbal formula Jakyakgamcho-tang (JYGCT; Paeonia lactiflora and Glycyrrhiza uralensis) has been used for treatment of backache, muscle pain, acute abdominal pain, neuralgia, bronchial asthma, and painful peripheral neuropathy in Oriental medicine. We report on our experiments using the HaCaT human keratinocyte cell line showing that a traditional herbal formula JYGCT has inhibitory effects on inflammatory responses in skin. Stimulation with tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) caused a significant increase in the production of the following chemokines: thymus- and activation-regulated chemokine (TARC)/CCL17; macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8) in HaCaT cells. By contrast, treatment with JYGCT extract significantly reduced the production of TARC, MDC, RANTES, and IL-8, but caused no cytotoxicity, compared with TNF-α and IFN-γ-treated control cells. Consistently, JYGCT extract downregulated the mRNA expression of TARC, MDC, RANTES, and IL-8 induced by TNF-α and IFN-γ in a dose-dependent manner. In addition, TNF-α and IFN-γ markedly increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and the nuclear translocation of nuclear factor kappa B (NF-κB) in HaCaT cells. By contrast, TNF-α and IFN-γ-induced activation of STAT1 and NF-κB activation was inhibited by JYGCT treatment in a dose-dependent manner. Our data indicate that JYGCT attenuates TNF-α and IFN-γ-mediated chemokine production by targeting the STAT1 and NF-κB signalling in keratinocytes. Our findings suggest that JYGCT has potential as a therapeutic drug candidate for the treatment of inflammatory skin diseases. PMID:25765840

  14. Phytoestrogens mediated anti-inflammatory effect through suppression of IRF-1 and pSTAT1 expressions in lipopolysaccharide-activated microglia.

    PubMed

    Jantaratnotai, Nattinee; Utaisincharoen, Pongsak; Sanvarinda, Pimtip; Thampithak, Anusorn; Sanvarinda, Yupin

    2013-10-01

    Microglial activation has been implicated in various neurological disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and HIV encephalopathy. Phytoestrogens have been shown to be neuroprotective in neurotoxicity models; however, their effect on microglia has not been well established. In the current study, we report that the soy phytoestrogens, genistein, daidzein, and coumestrol, decreased nitric oxide (NO) production induced by lipopolysaccharide (LPS) in the rat microglial cell line (HAPI). The levels of inducible NO synthase (iNOS) mRNA and protein expression were also reduced. Transcription factors known to govern iNOS expression including interferon regulatory factor-1 (IRF-1) and phosphorylated STAT1 were down regulated. These observations explain, at least in part, the inhibitory effect of phytoestrogens on NO production. The levels of monocyte chemoattractant protein-1 and interleukin-6 mRNA, proinflammatory chemokine and cytokine associated with various neurological disorders, were also reduced following LPS stimulation when HAPI cells were pretreated with phytoestrogens. Hence, genistein, daidzein, and coumestrol could serve as anti-inflammatory agents and may have beneficial effects in the treatment of neurodegenerative diseases.

  15. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  16. A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71.

    PubMed

    Liou, An-Ting; Wu, Szu-Yao; Liao, Chun-Che; Chang, Ya-Shu; Chang, Chih-Shin; Shih, Chiaho

    2016-01-01

    Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis. PMID:27499235

  17. A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71

    PubMed Central

    Liou, An-Ting; Wu, Szu-Yao; Liao, Chun-Che; Chang, Ya-Shu; Chang, Chih-Shin; Shih, Chiaho

    2016-01-01

    Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2+/+/stat-1−/−) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis. PMID:27499235

  18. A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71.

    PubMed

    Liou, An-Ting; Wu, Szu-Yao; Liao, Chun-Che; Chang, Ya-Shu; Chang, Chih-Shin; Shih, Chiaho

    2016-01-01

    Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis.

  19. Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1.

    PubMed

    Rahman, M Jubayer; Rahir, Gwendoline; Dong, Matthew B; Zhao, Yongge; Rodrigues, Kameron B; Hotta-Iwamura, Chie; Chen, Ye; Guerrero, Alan; Tarbell, Kristin V

    2016-03-01

    Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/β receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/β receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response. PMID:26826238

  20. MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

    PubMed Central

    Szpikowska, B. K.; Swiderek, K. M.; Sherman, M. A.; Mas, M. T.

    1998-01-01

    The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins. PMID:9684884

  1. Upregulation of the Suppressors of Cytokine Signaling 1 and 3 Is Associated with Arrest of Phosphorylated-STAT1 Nuclear Importation and Reduced Innate Response in Denguevirus-Infected Macrophages.

    PubMed

    Estrada-Jiménez, Tania; Millán-Pérez Peña, Lourdes; Flores-Mendoza, Lilian; Sedeño-Monge, Virginia; Santos-López, Gerardo; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Terán-Cabanillas, Eli; Hernández, Jesus; Herrera-Camacho, Irma; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio

    2016-03-01

    To clarify whether the suppressors of cytokine signaling (SOCS) are associated with denguevirus (DENV) evasion of the antiviral response, we analyzed the expression kinetics of SOCS1 and SOCS3 and of the antiviral genes MxA and OAS during DENV infection of U937 macrophages that were or not treated with interferon (IFN)-α. DENV infection produced a viral titer three times higher in untreated than in IFN-α-treated cells (p < 0.001 at 72 h postinfection [p.i.]). Partial inhibition of DENV replication was associated with reduced expression of MxA and OAS antiviral genes as well as higher SOCS1 and SOCS3 expression in DENV-infected cells than in cells treated only with IFN-α. Complete loss of phosphorylated-signal transducer and activator of transcription (p-STAT)2 and reduced nuclear importation of p-STAT1 were observed in DENV-infected cells compared to IFN-α treatment that induced p-STAT1 and p-STAT2. Our data thus suggest that overexpression of SOCS1 and SOCS3 induced by DENV infection leads to impairment of antiviral response through the inhibition of STAT functionality.

  2. Cathelicidin-BF suppresses intestinal inflammation by inhibiting the nuclear factor-κB signaling pathway and enhancing the phagocytosis of immune cells via STAT-1 in weanling piglets.

    PubMed

    Yi, Hongbo; Yu, Caihua; Zhang, Haiwen; Song, Deguang; Jiang, Denghu; Du, Huahua; Wang, Yizhen

    2015-09-01

    The severity of intestinal inflammation in mammals can be profoundly impacted by weaning stress. Cathelicidins protect intestinal homeostasis by not only directly killing bacteria but also immune regulators. Here, we investigated the effects of cathelicidin-BF (C-BF) derived from the snake venoms of Bungarus fasciatus on weaning stress and intestinal inflammation and examined the mechanisms by which C-BF modulates intestinal immune responses in weanling piglets. We found that C-BF treatment significantly increased performance and reduced the diarrheal index in weanling piglets. Serum IL-6, IL-22 and TNF-α production was decreased by C-BF treatment. We demonstrated that C-BF inhibited the expression of the inflammatory cytokines TNF-α, IL-6 and IL-8 but increased the expression of the anti-inflammatory cytokine IL-10 in the intestine. We also demonstrated that C-BF suppressed inflammation by down-regulating the nuclear factor-κB (NF-κB) signaling pathway in the intestine and in LPS-induced macrophages in vitro. However, C-BF significantly induced the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) to enhance the phagocytosis of macrophages when inflammation was suppressed. In summary, our study demonstrated that C-BF suppressed intestinal inflammation by down-regulating the NF-κB signaling pathway and enhancing the phagocytosis of immune cells by activating STAT-1 during weaning.

  3. Arginine residues within the DNA binding domain of STAT3 promote intracellular shuttling and phosphorylation of STAT3.

    PubMed

    Ginter, Torsten; Fahrer, Jörg; Kröhnert, Ulrike; Fetz, Verena; Garrone, Alessio; Stauber, Roland H; Reichardt, Werner; Müller-Newen, Gerhard; Kosan, Christian; Heinzel, Thorsten; Krämer, Oliver H

    2014-08-01

    Acetylation-dependent inactivation of STAT1 can be mimicked by the exchange of its lysine residues K410 and K413 to glutamine residues. STAT3 harbors non-acetylatable arginine moieties at the corresponding sites R414 and R417. It is unclear whether the mutation of these sites to glutamine residues antagonizes STAT3 activation. Here, we show that an arginine-glutamine-exchange at the STAT3 moieties R414 and R417 (R414Q and R417Q) reduces cytokine-dependent tyrosine phosphorylation of STAT3. This inhibitory effect can be partially rescued by phosphatase inhibition. In addition, the R414Q and R417Q mutations enhance the nuclear accumulation of unphosphorylated STAT3. STAT3 R414Q and STAT3 R417Q show a reduced response to cytokine stimulation emanating from the plasma membrane. Moreover, these STAT3 mutants have no direct inhibitory effect on the cytokine-induced activation of STAT1/STAT3-mediated gene expression. Since the mutations R414Q and R417Q reside within the STAT3 DNA binding domain (DBD), the STAT3 R414Q and R417Q mutants also lack intrinsic activity as transcription factors. Furthermore, in contrast to wild-type STAT3 they cannot compensate for a loss of STAT1 and they cannot promote STAT1/STAT3-dependent transcriptional activation. We further analyzed a STAT3 arginine-lysine-exchange mutant (R414K/R417K). This molecule mimics corresponding lysine residues found within the DBD of STAT1. Compared to wild-type STAT3, the STAT3 R414K/R417K mutant shows attenuated tyrosine phosphorylation and it is a less active transcription factor. In addition, STAT3 R414K/R417K is not activated by deacetylase inhibition. On the other hand, C-terminal acetylation of STAT3 is intact in STAT3 R414K/R417K. Our results suggest that the exchange of amino acid residues within the DBDs of STAT1/STAT3 affects their phosphorylation as well as their intracellular shuttling. PMID:24721162

  4. Bilirubin binding with liver cystatin induced structural and functional changes.

    PubMed

    Mustafa, Mir Faisal; Bano, Bilqees

    2014-05-01

    Cysteine proteinases and their inhibitors play a significant role in the proteolytic environment of the cells. Inhibitors of cysteine proteinases regulate the activity of these enzymes helping in checking the degdration activity of cathepsins. The bilirubin secreated by liver cells can bind to cystatin present in the liver resulting in its functional inactivation, which may further lead to the increase in cathepsins level causing liver cirrhosis. In case of some pathophysiological conditions excess bilirubin gets accumulated e.g. in presence of Fasciola hepatica (liver fluke) in mammals and humans, leading to liver cirrhosis and possibly jaundice or normal blockade of bile duct causing increased level of bilirubin in blood. Protease-cystatin imbalance causes disease progression. In the present study, Bilirubin (BR) and liver cystatin interaction was studied to explore the cystatin inactivation and structural alteration. The binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of BR with buffalo liver cystatin (BLC). Stern-Volmer analysis of BR-BLC system indicates the presence of static component in the quenching mechanism and the number of binding sites to be close to 1. The fluorescence data proved that the fluorescence quenching of liver cystatin by BR was the result of BR-cystatin complex formation. FTIR analysis of BR-Cystatin complex revealed change in the secondary structure due to perturbation in the microenvironment further confirmed by the decreased caseinolytic activity of BLC against papain. Fluorescence measurements also revealed quenching of fluorescence and shift in peak at different time intervals and at varying pH values. Photo-illumination of BR-cystatin complex causes change in the surrounding environment of liver cystatin as indicated by red-shift. The binding constant for BR-BLC complex was found to be 9.279 × 10(4) M(-1). The cystatin binding with

  5. Deficient Phosphorylation of Stat1 in Leukocytes Identifies Neutralizing Antibodies in Multiple Sclerosis Patients Treated with Interferon-Beta

    PubMed Central

    Gavasso, Sonia; Mosleth, Ellen Faergestad; Marøy, Tove; Jørgensen, Katarina; Nakkestad, Hanne-Linda; Gjertsen, Bjørn-Tore; Myhr, Kjell-Morten; Vedeler, Christian

    2014-01-01

    Background Anti interferon-beta (IFN-β) neutralizing antibodies (NAb) affect efficacy of treatment of multiple sclerosis patients, but exactly when the detrimental effects of NAbs offset therapeutic efficacy is debated. Quantification of intracellular pathway-specific phosphorylation by phospho-specific flow cytometry (phosphoflow) is a promising tool for evaluation of these effects in primary immune cells from treated patients at the single-cell level. Method Samples for phosphoflow and gene expression changes were collected before administration of IFN-β and at four, six, and eight hours thereafter. Patients were NAb negative (n = 3) or were NAb positive with low/medium (n = 1) or high (n = 2) NAb titers. Levels of phosphorylation of six Stat transcription factors (pStat) in seven cell subtypes and expression levels of 71 pathway-specific genes in whole blood were measured. The data was subjected to principal component analysis (PCA), fifty-fifty MANOVA, ANOVA, and partial least square regression (PLSR). Results PCA of pStat levels clustered patients according to NAb class independently of time. PCA of gene expression data clustered patients according to NAb class but was affected by time and treatment. In the fifty-fifty MANOVA, NAb class was significant for both pStat levels and gene expression data. The ANOVA identified pStat1 protein in several cell subtypes as significantly affected by NAb class. The best fitting model for NAb prediction based on PLSR included pStat1 in monocytes, T cells, or lymphocytes and pStat3 in monocytes (r = 0.97). Gene expression data were slightly less predictive of NAb titers. Conclusion Based on this proof of concept study, we hypothesize that NAb effects can be monitored by evaluation of a single biomarker, pStat1, in either monocytes or T cells by phosphoflow directly after IFN-β administration. The method will significantly reduce cost relative to labor intensive in vitro methods and offers a patient

  6. Development of Th17-associated interstitial kidney inflammation in lupus-prone mice lacking the gene encoding Signal Transduction and Activator of Transcription-1 (STAT-1)

    PubMed Central

    Yiu, Gloria; Rasmussen, Tue Kruse; Ajami, Bahareh; Haddon, David J.; Chu, Alvina D.; Tangsombatvisit, Stephanie; Haynes, Winston A.; Diep, Vivian; Steinman, Larry; Faix, James; Utz, Paul J.

    2016-01-01

    Type I interferon (IFN-I) signaling is a central pathogenic pathway in Systemic Lupus Erythematosus (SLE), and therapeutics targeting IFN-I signaling are in development. Multiple proteins with overlapping function participate in IFN signaling, but the signaling events downstream of receptor engagement are unclear. We employed highly-multiplexed assays to characterize autoantibody production, cytokine/chemokine profiles, and Signal Transduction and Activators of Transcription (STAT) phosphorylation to investigate the individual roles of IFNAR2, IRF9 and STAT1 in MRL/lpr (lpr) mice. Surprisingly, we found that Stat1−/−, but not Irf9−/− or Ifnar2−/− mice, developed interstitial nephritis characterized by infiltration with RORγT+ lymphocytes, macrophages and eosinophils. Despite pronounced interstitial kidney disease and abnormal kidney function, Stat1−/− mice had decreased proteinuria, glomerulonephritis and autoantibody production. Phospho-specific flow cytometry (phosphoflow) revealed shunting of STAT phosphorylation from STAT1 to STAT3/4. In summary, we describe unique contributions of STAT1 to pathology in different kidney compartments, and provide novel insight into tubulointerstitial nephritis (TIN) a poorly understood complication that predicts end-stage kidney disease in SLE patients. PMID:26636548

  7. Induced circularly polarized luminescence arising from anion or protein binding to racemic emissive lanthanide complexes

    NASA Astrophysics Data System (ADS)

    Carr, Rachel; Puckrin, Robert; McMahon, Brian K.; Pal, Robert; Parker, David; Pålsson, Lars-Olof

    2014-06-01

    A circularly polarized luminescence (CPL) spectrometer has been built and used to study the binding interaction of lactate and four different proteins with racemic EuIII and TbIII complexes in aqueous solution. Lactate binding gives rise to strong induced CPL spectra, and the observed emission dissymmetry factors vary linearly with enantiomeric composition. Particularly strong induced TbIII CPL also characterizes the binding interaction of alpha-1-acid glycoprotein with a dissociation constant, Kd, of 2.5 μM.

  8. A STAT1-gain-of-function mutation causing Th17 deficiency with chronic mucocutaneous candidiasis, psoriasiform hyperkeratosis and dermatophytosis.

    PubMed

    Nielsen, Jakob; Kofod-Olsen, Emil; Spaun, Eva; Larsen, Carsten S; Christiansen, Mette; Mogensen, Trine Hyrup

    2015-01-01

    During recent years, inborn errors of human IL-17 immunity have been demonstrated to underlie primary immunodeficiencies with chronic mucocutaneous candidiasis (CMC). Various defects in receptors responsible for sensing of Candida albicans or downstream signalling to IL-17 may lead to susceptibility to Candida infection. While CMC is common in patients with profound T cell immunodeficiencies, CMC is also recognised as part of other immunodeficiencies in syndromic CMC, or as relatively isolated CMC disease. We describe a 40-year-old woman with a clinical picture involving cutaneous bacterial abscesses, chronic oral candidiasis and extensive dermatophytic infection of the feet. By whole exome sequencing, we identified a STAT1-gain-of-function mutation. Moreover, the patient's peripheral blood mononuclear cells displayed severely impaired Th17 responses. The patient was treated with antifungals and prophylactic antibiotics, which led to resolution of the infection. We discuss the current knowledge within the field of Th17 deficiency and the pathogenesis and treatment of CMC.

  9. Fragment growing induces conformational changes in acetylcholine-binding protein: a structural and thermodynamic analysis.

    PubMed

    Edink, Ewald; Rucktooa, Prakash; Retra, Kim; Akdemir, Atilla; Nahar, Tariq; Zuiderveld, Obbe; van Elk, René; Janssen, Elwin; van Nierop, Pim; van Muijlwijk-Koezen, Jacqueline; Smit, August B; Sixma, Titia K; Leurs, Rob; de Esch, Iwan J P

    2011-04-13

    Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP). This protein is a soluble homologue of the ligand binding domain (LBD) of Cys-loop receptors. The fragment optimization was monitored with X-ray structures of ligand complexes and systematic thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC). Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles, are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and fragment-based discovery approaches. PMID:21322593

  10. Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

    PubMed

    Yamaguchi, Hiroto; Miwa, Yukiko; Kasa, Miyuki; Kitano, Ken; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-09-01

    Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability. PMID:16891330

  11. Stat1-Deficient Mice Are Not an Appropriate Model for Efficacy Testing of Recombinant Vesicular Stomatitis Virus–Based Filovirus Vaccines

    PubMed Central

    Marzi, Andrea; Kercher, Lisa; Marceau, Joshua; York, Anthony; Callsion, Julie; Gardner, Donald J.; Geisbert, Thomas W.; Feldmann, Heinz

    2015-01-01

    Stat1−/− mice lack a response to interferon α, β, and γ, allowing for replication of nonadapted wild-type (wt) Ebolavirus and Marburgvirus. We sought to establish a mouse model for efficacy testing of live attenuated recombinant vesicular stomatitis virus (rVSV)–based filovirus vaccine vectors using wt Ebolavirus and Marburgvirus challenge strains. While infection of immunocompetent mice with different rVSV-based filovirus vectors did not cause disease, infection of Stat1−/− mice with the same vectors resulted in systemic infection and lethal outcome for the majority of tested rVSVs. Despite differences in viral loads, organ tropism was remarkably similar between rVSV filovirus vaccine vectors and rVSVwt, with the exception of the brain. In conclusion, Stat1−/− mice are not an appropriate immunocompromised mouse model for efficacy testing of live attenuated, replication-competent rVSV vaccine vectors. PMID:26022440

  12. An oxygen-induced but protein F-independent fibronectin-binding pathway in Streptococcus pyogenes.

    PubMed Central

    Lee, J Y; Caparon, M

    1996-01-01

    Protein F is an important fibronectin-binding adhesin of Streptococcus pyogenes (group A streptococcus). However, all previous analyses of protein F have been conducted in a mutant strain which expresses protein F under anaerobic conditions nonpermissive for expression in other strains. In this study, we have examined the fibronectin-binding properties of several protein F-deficient mutants cultured under aerobic conditions and have identified a second pathway for binding fibronectin. Unlike the case with protein F, exposure to an aerobic environment does not induce transcription of a new gene product. Rather, O2 is apparently required for the modification of a protease-resistant cell surface component into a binding-component form. Modification occurred preferentially at a pH of 6.0 or less, and the binding of the modified component to fibronectin required Zn2+. The oxidizing agent Fe(CN)6 could be substituted for O2 and stimulated expression of binding activity under O2-limiting conditions. Streptococcal fibronectin binding mediated by this pathway but not by protein F could be inhibited by laminin and by streptococcal lipoteichoic acid, a molecule previously implicated as the streptococcal adhesin for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism to basement membrane. By using differential inhibition, analyses of binding to non-protein F mutant strains demonstrated that the total level of fibronectin bound under aerobic conditions reflects contributions from both pathways. Because of its dependence on Zn2+, an oxidant, and pH, this binding activity has been designated the ZOP binding pathway. PMID:8550185

  13. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  14. Phospholipid-binding proteins differ in their capacity to induce autoantibodies and murine systemic lupus erythematosus.

    PubMed

    Levine, J S; Subang, R; Setty, S; Cabrera, J; Laplante, P; Fritzler, M J; Rauch, J

    2014-07-01

    We have previously shown that immunization of nonautoimmune mice with the phospholipid-binding protein β2-glycoprotein I (β2GPI), in combination with lipopolysaccharide (LPS), induces a murine model of systemic lupus erythematosus (SLE), with sequential emergence of autoantibodies and glomerulonephritis. Here, we determine whether the paradigm for induction of murine SLE extends to other phospholipid-binding proteins. Mice were immunized with a phospholipid-binding protein (prothrombin (PT), protein S, or β2GPI), or a nonphospholipid-binding protein (glu-plasminogen), in the presence of LPS. The breadth and degree of the autoantibody response, and the frequency of glomerulonephritis, varied among the three proteins, with β2GPI being the most effective in inducing SLE-like disease. The phospholipid-binding proteins also differed in the pattern of serum cytokines they elicited. The most apparent difference between β2GPI and the other phospholipid-binding proteins was in their ability to bind to LPS: β2GPI bound to LPS, while PT and protein S did not. Our data suggest that binding to phospholipid(s) is a necessary, but not sufficient, condition for full induction of murine SLE. We propose that other properties, such as physiologic function, avidity for anionic phospholipids, and degree of interaction with other cell surface and/or circulating molecules (particularly LPS) may determine the range and severity of disease.

  15. Amino acid composition of cadmium-binding protein induced in a marine diatom

    SciTech Connect

    Maita, Y.; Kawaguchi, S. )

    1989-09-01

    Organisms living in environments polluted with heavy metals develop tolerance against these contaminants. The tolerance has been attributed to the ability to synthesize metal binding substances. These recent findings imply metal binding complexes from animals and plants, although having very similar functional properties, may have entirely different amino acid compositions. Researchers reported that cadystin from fission yeast, Schizosaccharomyces pombe was composed of only glutamic acid, cysteine, and glycine. A year later, a heavy metal binding substance was isolated from Rauwolfia serpetina which contains only Glu, Cys, and Gly. Heavy metal binding complexes isolated from the water hyacinth and morning glory Datura innoxia also showed an amino acid composition similar to cadystin or phytochelatin. In this study, the cadmium binding protein induced in the marine diatom, Phaeodactylum tricornutum, was isolated and purified and its amino acid composition determined.

  16. Anthramycin binding to deoxyribonucleic acid-mitomycin C complexes. Evidence for drug-induced deoxyribonucleic acid conformational change and cooperativity in mitomycin C binding.

    PubMed

    Kaplan, D J; Hurley, L H

    1981-12-22

    Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change. PMID:6798992

  17. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.

    PubMed

    Franceschi, Valentina; Parker, Scott; Jacca, Sarah; Crump, Ryan W; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D; Wong, Scott W; Buller, Mark R; Donofrio, Gaetano

    2015-06-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  18. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

    PubMed Central

    Crump, Ryan W.; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D.; Wong, Scott W.; Buller, Mark R.; Donofrio, Gaetano

    2015-01-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  19. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.

    PubMed

    Franceschi, Valentina; Parker, Scott; Jacca, Sarah; Crump, Ryan W; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D; Wong, Scott W; Buller, Mark R; Donofrio, Gaetano

    2015-06-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  20. IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism*

    PubMed Central

    Kopach, Pavel; Lockatell, Virginia; Pickering, Edward M.; Haskell, Ronald E.; Anderson, Richard D.; Hasday, Jeffrey D.; Todd, Nevins W.; Luzina, Irina G.; Atamas, Sergei P.

    2014-01-01

    IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion. PMID:24619410

  1. Flexibility in the inducer binding region is crucial for allostery in the Escherichia coli lactose repressor.

    PubMed

    Xu, Jia; Matthews, Kathleen S

    2009-06-01

    Lactose repressor protein (LacI) utilizes an allosteric mechanism to regulate transcription in Escherichia coli, and the transition between inducer- and operator-bound states has been simulated by targeted molecular dynamics (TMD). The side chains of amino acids 149 and 193 interact and were predicted by TMD simulation to play a critical role in the early stages of the LacI conformational change. D149 contacts IPTG directly, and variations at this site provide the opportunity to dissect its role in inducer binding and signal transduction. Single mutants at D149 or S193 exhibit a minimal change in operator binding, and alterations in inducer binding parallel changes in operator release, indicating normal allosteric response. The observation that the double mutant D149A/S193A exhibits wild-type properties excludes the requirement for inter-residue hydrogen bond formation in the allosteric response. The double mutant D149C/S193C purified from cell extracts shows decreased sensitivity to inducer binding while retaining wild-type binding affinities and kinetic constants for both operator and inducer. By manipulating cysteine oxidation, we show that the more reduced state of D149C/S193C responds to inducer more like the wild-type protein, whereas the more oxidized state displays diminished inducer sensitivity. These features of D149C/S193C indicate that the novel disulfide bond formed in this mutant impedes the allosteric transition, consistent with the role of this region predicted by TMD simulation. Together, these results establish the requirement for flexibility in the spatial relationship between D149 and S193 rather than a specific D149-S193 interaction in the LacI allosteric response to inducer. PMID:19368358

  2. Toxoplasma Effector Recruits the Mi-2/NuRD Complex to Repress STAT1 Transcription and Block IFN-γ-Dependent Gene Expression.

    PubMed

    Olias, Philipp; Etheridge, Ronald D; Zhang, Yong; Holtzman, Michael J; Sibley, L David

    2016-07-13

    Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses. PMID:27414498

  3. Gene array analysis of a rat model of liver transplant tolerance identifies increased complement C3 and the STAT-1/IRF-1 pathway during tolerance induction.

    PubMed

    Cordoba, Shaun P; Wang, Chuanmin; Williams, Rohan; Li, Jian; Smit, Lynn; Sharland, Alexandra; Allen, Richard; McCaughan, Geoffrey; Bishop, Alex

    2006-04-01

    This study aimed to define the molecular mechanism during induction of spontaneous liver transplant tolerance using microarrays and to focus on molecular pathways associated with tolerance by meta-analysis with published studies. The differences in the early immune response between PVG to DA liver transplant recipients that are spontaneously tolerant (TOL) and PVG to Lewis liver transplants that reject (REJ) were examined. Spleens from TOL and REJ on days 1 and 3 were compared by 2 color microarray. Forty-six of 199 genes differentially expressed between TOL and REJ had an immunological function. More immune genes were increased in TOL vs. REJ on day 1, including STAT-1, IRF-1 and complement C3. Differential expression of selected genes was confirmed by quantitative RT-PCR. The results were compared to two published high-throughput studies of rat liver transplant tolerance and showed that C3 was increased in all three models, while STAT-1 and IRF-1 were increased in two models. The early increases in immune genes in TOL confirmed previous reports of an active early immune response in TOL. In conclusion, the increase in STAT-1, IRF-1 and complement component C3 in several models of liver transplant tolerance suggests that the STAT-1/IRF-1 apoptotic pathway and C3 may be involved in the tolerogenic mechanism.

  4. Ligand Binding to the Androgen Receptor Induces Conformational Changes That Regulate Phosphatase Interactions▿

    PubMed Central

    Yang, Chun-Song; Xin, Hong-Wu; Kelley, Joshua B.; Spencer, Adam; Brautigan, David L.; Paschal, Bryce M.

    2007-01-01

    We describe a mechanism for protein phosphatase 2A (PP2A) targeting to the androgen receptor (AR) and provide insight into the more general issue of kinase and phosphatase interactions with AR. Simian virus 40 (SV40) small t antigen (ST) binding to N-terminal HEAT repeats in the PP2A A subunit induces structural changes transduced to C-terminal HEAT repeats. This enables the C-terminal HEAT repeats in the PP2A A subunit, including HEAT repeat 13, to discriminate between androgen- and androgen antagonist-induced AR conformations. The PP2A-AR interaction was used to show that an AR mutant in prostate cancer cells (T877A) is activated by multiple ligands without acquiring the same conformation as that induced by androgen. The correlation between androgen binding to AR and increased phosphorylation of the activation function 1 (AF-1) region implies that changes in AR conformation or chaperone composition are causal to kinase access to phosphorylation sites. However, AF-1 phosphorylation sites are kinase accessible prior to androgen binding. This suggests that androgens can enhance the phosphorylation state of AR either by negatively regulating the ability of the ligand-binding domain to bind phosphatases or by inducing an AR conformation that is resistant to phosphatase action. SV40 ST subverts this mechanism by promoting the direct transfer of PP2A onto androgen-bound AR, resulting in multisite dephosphorylation. PMID:17325038

  5. Pathogenesis and Immune Response of Crimean-Congo Hemorrhagic Fever Virus in a STAT-1 Knockout Mouse Model▿ †

    PubMed Central

    Bente, Dennis A.; Alimonti, Judie B.; Shieh, Wun-Ju; Camus, Gaëlle; Ströher, Ute; Zaki, Sherif; Jones, Steven M.

    2010-01-01

    Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) causes a severe hemorrhagic syndrome in humans but not in its vertebrate animal hosts. The pathogenesis of the disease is largely not understood due to the lack of an animal model. Laboratory animals typically show no overt signs of disease. Here, we describe a new small-animal model to study CCHFV pathogenesis that manifests clinical disease, similar to that seen in humans, without adaptation of the virus to the host. Our studies revealed that mice deficient in the STAT-1 signaling molecule were highly susceptible to infection, succumbing within 3 to 5 days. After CCHFV challenge, mice exhibited fever, leukopenia, thrombocytopenia, and highly elevated liver enzymes. Rapid viremic dissemination and extensive replication in visceral organs, mainly in liver and spleen, were associated with prominent histopathologic changes in these organs. Dramatically elevated proinflammatory cytokine levels were detected in the blood of the animals, suggestive of a cytokine storm. Immunologic analysis revealed delayed immune cell activation and intensive lymphocyte depletion. Furthermore, this study also demonstrated that ribavirin, a suggested treatment in human cases, protects mice from lethal CCHFV challenge. In conclusion, our data demonstrate that the interferon response is crucial in controlling CCHFV replication in this model, and this is the first study that offers an in-depth in vivo analysis of CCHFV pathophysiology. This new mouse model exhibits key features of fatal human CCHF, proves useful for the testing of therapeutic strategies, and can be used to study virus attenuation. PMID:20739514

  6. Elevated levels of STAT1 in Fanconi anemia group A lymphoblasts correlate with the cells’ sensitivity to DNA interstrand crosslinking drugs

    PubMed Central

    Prieto-Remón, Inés; Sánchez-Carrera, Dámaso; López-Duarte, Mónica; Richard, Carlos; Pipaón, Carlos

    2013-01-01

    Progressive bone marrow failure starting in the first decade of life is one of the main characteristics of Fanconi anemia. Along with the bone marrow failure, this pathology is characterized by congenital malformations, endocrine dysfunction and an extraordinary predisposition to develop cancer. The fact that hematopoietic progenitor cells from subjects with Fanconi anemia are sensitive to both DNA-interstrand crosslinking agents and inflammatory cytokines, which are aberrantly overproduced in these patients, has led to different explanations for the causes of the bone marrow failure. We analyzed STAT1 expression in lymphoblastoid cell lines derived from patients with Fanconi anemia group A and correlated this with aspects of the Fanconi anemia phenotype such as sensitivity to genotoxic agents or to inhibitory cytokines. We provide evidence of overexpression of STAT1 in FANCA-deficient cells which has both transcriptional and post-translational components, and is related to the constitutive activation of ERK in Fanconi anemia group A cells, since it can be reverted by treatment with U0126. STAT1 phosphorylation was not defective in the lymphoblasts, so these cells accumulated higher levels of active STAT1 in response to interferon gamma, probably in relation to their greater sensitivity to this cytokine. On the other hand, inhibition of STAT1 by genetic or chemical means reverted the hypersensitivity of Fanconi anemia group A lymphoblasts to DNA interstrand crosslinking agents. Our data provide an explanation for the mixed sensitivity of Fanconi anemia group A cells to both genotoxic stress and inflammatory cytokines and indicate new targets for the treatment of bone marrow failure in these patients. PMID:23585528

  7. Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element.

    PubMed Central

    Ohme-Takagi, M; Shinshi, H

    1995-01-01

    We demonstrated that the GCC box, which is an 11-bp sequence (TAAGAGCCGCC) conserved in the 5' upstream region of ethylene-inducible pathogenesis-related protein genes in Nicotiana spp and in some other plants, is the sequence that is essential for ethylene responsiveness when incorporated into a heterologous promoter. Competitive gel retardation assays showed DNA binding activities to be specific to the GCC box sequence in tobacco nuclear extracts. Four different cDNAs encoding DNA binding proteins specific for the GCC box sequence were isolated, and their products were designated ethylene-responsive element binding proteins (EREBPs). The deduced amino acid sequences of EREBPs exhibited no homology with those of known DNA binding proteins or transcription factors; neither did the deduced proteins contain a basic leucine zipper or zinc finger motif. The DNA binding domain was identified within a region of 59 amino acid residues that was common to all four deduced EREBPs. Regions highly homologous to the DNA binding domain of EREBPs were found in proteins deduced from the cDNAs of various plants, suggesting that this domain is evolutionarily conserved in plants. RNA gel blot analysis revealed that accumulation of mRNAs for EREBPs was induced by ethylene, but individual EREBPs exhibited different patterns of expression. PMID:7756828

  8. Enhancement of binding kinetics on affinity substrates by laser point heating induced transport.

    PubMed

    Wang, Bu; Cheng, Xuanhong

    2016-03-01

    Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used the COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates is quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces the reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predictable binding patterns. This optothermal strategy holds promise to improve the time-response and sensitivity of biosensors and microarrays. PMID:26898559

  9. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  10. Structural changes in cytochrome c oxidase induced by binding of sodium and calcium ions: an ATR-FTIR study.

    PubMed

    Maréchal, Amandine; Iwaki, Masayo; Rich, Peter R

    2013-04-17

    Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the binding of Na(+) and Ca(2+)cations to bovine cytochrome c oxidase in its fully oxidized and partially reduced, cyanide-ligated (a(2+)a3(3+)-CN) (mixed valence) forms. These ions induced distinctly different IR binding spectra, indicating that the induced structural changes are different. Despite this, their binding spectra were mutually exclusive, confirming their known competitive binding behavior. Dissociation constants for Na(+) and Ca(2+) with the oxidized enzyme were 1.2 mM and 11 μM, respectively and Na(+) binding appeared to involve cooperative binding of two Na(+). Ca(2+) binding induced a large IR spectrum, with prominent amide I/II polypeptide changes, bandshifts assigned to carboxylate and an arginine, and a number of bandshifts of heme a. The Na(+)-induced binding spectrum showed much weaker amide I/II and heme a changes but had similar shifts assignable to carboxylate and arginine residues. Yeast CcO also displayed a calcium-induced IR and UV/visible binding spectra, though of lower intensities. This was attributed to the difficulty in fully depleting Ca(2+) from its binding site, as has been found with bacterial CcOs. The implications of Ca(2+)/Na(+) ion binding are discussed in terms of structure and possible modulation of core catalytic function. PMID:23537388

  11. Ligand-induced conformational changes in a thermophilic ribose-binding protein

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2009-05-21

    Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (appTm value is 108 C) than the mesophilic Escherichia coli homolog (ecRBP) (appTm value is 56 C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different

  12. DNA damage–inducible SUMOylation of HERC2 promotes RNF8 binding via a novel SUMO-binding Zinc finger

    PubMed Central

    Danielsen, Jannie Rendtlew; Povlsen, Lou Klitgaard; Villumsen, Bine Hare; Streicher, Werner; Nilsson, Jakob; Wikström, Mats; Bekker-Jensen, Simon

    2012-01-01

    Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by the RNF8/RNF168/HERC2 ubiquitin ligases facilitates restoration of genome integrity by licensing chromatin to concentrate genome caretaker proteins near the lesions. In parallel, SUMOylation of so-far elusive upstream DSB regulators is also required for execution of this ubiquitin-dependent chromatin response. We show that HERC2 and RNF168 are novel DNA damage–dependent SUMOylation targets in human cells. In response to DSBs, both HERC2 and RNF168 were specifically modified with SUMO1 at DSB sites in a manner dependent on the SUMO E3 ligase PIAS4. SUMOylation of HERC2 was required for its DSB-induced association with RNF8 and for stabilizing the RNF8–Ubc13 complex. We also demonstrate that the ZZ Zinc finger in HERC2 defined a novel SUMO-specific binding module, which together with its concomitant SUMOylation and T4827 phosphorylation promoted binding to RNF8. Our findings provide novel insight into the regulatory complexity of how ubiquitylation and SUMOylation cooperate to orchestrate protein interactions with DSB repair foci. PMID:22508508

  13. Proposed Standards for Variable Harmonization Documentation and Referencing: A Case Study Using QuickCharmStats 1.1.

    PubMed

    Winters, Kristi; Netscher, Sebastian

    2016-01-01

    Comparative statistical analyses often require data harmonization, yet the social sciences do not have clear operationalization frameworks that guide and homogenize variable coding decisions across disciplines. When faced with a need to harmonize variables researchers often look for guidance from various international studies that employ output harmonization, such as the Comparative Survey of Election Studies, which offer recoding structures for the same variable (e.g. marital status). More problematically there are no agreed documentation standards or journal requirements for reporting variable harmonization to facilitate a transparent replication process. We propose a conceptual and data-driven digital solution that creates harmonization documentation standards for publication and scholarly citation: QuickCharmStats 1.1. It is free and open-source software that allows for the organizing, documenting and publishing of data harmonization projects. QuickCharmStats starts at the conceptual level and its workflow ends with a variable recording syntax. It is therefore flexible enough to reflect a variety of theoretical justifications for variable harmonization. Using the socio-demographic variable 'marital status', we demonstrate how the CharmStats workflow collates metadata while being guided by the scientific standards of transparency and replication. It encourages researchers to publish their harmonization work by providing researchers who complete the peer review process a permanent identifier. Those who contribute original data harmonization work to their discipline can now be credited through citations. Finally, we propose peer-review standards for harmonization documentation, describe a route to online publishing, and provide a referencing format to cite harmonization projects. Although CharmStats products are designed for social scientists our adherence to the scientific method ensures our products can be used by researchers across the sciences.

  14. Protein inhibitor of activated STAT-1 is downregulated in gastric cancer tissue and involved in cell metastasis.

    PubMed

    Chen, Ping; Zhao, Deshou; Sun, Yunwei; Huang, Liya; Zhang, Shuxian; Yuan, Yaozong

    2012-12-01

    Protein inhibitor of activated STAT-1 (PIAS1) is a novel modulator of the JAK/STAT signaling pathway that negatively regulates the inflammatory response. It has been also reported to be downregulated in a variety of human cancer cell lines. However, the role of PIAS1 in gastric cancer remains unclear. In this study, we investigated the prognostic value of PIAS1 expression and its regulated mechanisms in gastric cancer cell metastasis. Therefore, the expression of PIASI was explored in gastric cancer tissues and adjacent tissues of gastric cancer with 31 cases of patients, and the prognostic value was analyzed. In addition, the growth and invasion in SGC7901 cells were investigated in the restoration of PIAS1 expression with Ad5/F35-PIAS1 or Ad5/F35-vector or PBS treatment, and the activity of P38MAPK, P-P38MAPK, JNK/SAPK, P-JNK/SAPK, ERK and P-ERK, were detected by western blotting. The tumor migratory factors MMP-9, MMP-2 and ICAM-1 were analyzed by western blotting. The results demonstrated that 22 of 31 (70.9%) gastric cancer specimens showed low levels of PIAS1 expression from immunohistochemistry staining using tissue microarrays. Statistical analysis suggested that the downregulation of PIAS1 was significantly correlated with tumor staging. Furthermore, we found that the restoration of PIAS1 expression mediated by Ad5/F35 virus suppressed cell proliferation and invasion accompanied by the inhibition of P38MAPK and ERK protein expression and activity, but not JNK/SAPK protein. Notably, PIAS1 restoration with the transfection of Ad5/F35-PIAS1 robustly decreased the expression of tumor migratory factors including MMP-9, MMP-2 and ICAM-1 compared to Ad5/F35-vector. These data suggest that PIAS1 may function as a tumor suppressor to regulate gastric cancer cell metastasis by targeting the MAPK signaling pathway.

  15. Proposed Standards for Variable Harmonization Documentation and Referencing: A Case Study Using QuickCharmStats 1.1

    PubMed Central

    Winters, Kristi; Netscher, Sebastian

    2016-01-01

    Comparative statistical analyses often require data harmonization, yet the social sciences do not have clear operationalization frameworks that guide and homogenize variable coding decisions across disciplines. When faced with a need to harmonize variables researchers often look for guidance from various international studies that employ output harmonization, such as the Comparative Survey of Election Studies, which offer recoding structures for the same variable (e.g. marital status). More problematically there are no agreed documentation standards or journal requirements for reporting variable harmonization to facilitate a transparent replication process. We propose a conceptual and data-driven digital solution that creates harmonization documentation standards for publication and scholarly citation: QuickCharmStats 1.1. It is free and open-source software that allows for the organizing, documenting and publishing of data harmonization projects. QuickCharmStats starts at the conceptual level and its workflow ends with a variable recording syntax. It is therefore flexible enough to reflect a variety of theoretical justifications for variable harmonization. Using the socio-demographic variable ‘marital status’, we demonstrate how the CharmStats workflow collates metadata while being guided by the scientific standards of transparency and replication. It encourages researchers to publish their harmonization work by providing researchers who complete the peer review process a permanent identifier. Those who contribute original data harmonization work to their discipline can now be credited through citations. Finally, we propose peer-review standards for harmonization documentation, describe a route to online publishing, and provide a referencing format to cite harmonization projects. Although CharmStats products are designed for social scientists our adherence to the scientific method ensures our products can be used by researchers across the sciences. PMID

  16. Arctigenin protects cultured cortical neurons from glutamate-induced neurodegeneration by binding to kainate receptor.

    PubMed

    Jang, Young P; Kim, So R; Choi, Young H; Kim, Jinwoong; Kim, Sang G; Markelonis, George J; Oh, Tae H; Kim, Young C

    2002-04-15

    We previously reported that arctigenin, a lignan isolated from the bark of Torreya nucifera, showed significant neuroprotective activity against glutamate-induced toxicity in primary cultured rat cortical cells. In this study, the mode of action of arctigenin was investigated using primary cultures of rat cortical cells as an in vitro system. Arctigenin significantly attenuated glutamate-induced neurotoxicity when added prior to or after an excitotoxic glutamate challenge. The lignan protected cultured neuronal cells more selectively from neurotoxicity induced by kainic acid than by N-methyl-D-aspartate. The binding of [(3)H]-kainate to its receptors was significantly inhibited by arctigenin in a competitive manner. Furthermore, arctigenin directly scavenged free radicals generated by excess glutamate and successfully reduced the level of cellular peroxide in cultured neurons. These results suggest that arctigenin exerted significant neuroprotective effects on glutamate-injured primary cultures of rat cortical cells by directly binding to kainic acid receptors and partly scavenging of free radicals.

  17. Induced conformational changes upon Cd2+ binding at photosynthetic reaction centers

    PubMed Central

    Ishikita, Hiroshi; Knapp, Ernst-Walter

    2005-01-01

    Cd2+ binding at the bacterial photosynthetic reaction center (bRC) from Rhodobacter sphaeroides is known to inhibit proton transfer (PT) from bulk solvent to the secondary quinone QB. To elucidate this mechanism, we calculated the pKa for residues along the water channels connecting QB with the stromal side based on the crystal structures of WT-bRC and Cd2+-bound bRC. Upon Cd2+ binding, we observed the release of two protons from His-H126/128 at the Cd2+ binding site and significant pKa shifts for residues along the PT pathways. Remarkably, Asp-L213 near QB, which is proposed to play a significant role in PT, resulted in a decrease in pKa upon Cd2+ binding. The direct electrostatic influence of the Cd2+-positive charge on these pKa shifts was small. Instead, conformational changes of amino acid side chains induced electrostatically by Cd2+ binding were the main mechanism for these pKa shifts. The long-range electrostatic influence over ≈12 Å between Cd2+ and Asp-L213 is likely to originate from a set of Cd2+-induced successive reorientations of side chains (Asp-H124, His-H126, His-H128, Asp-H170, Glu-H173, Asp-M17, and Asp-L210), which propagate along the PT pathways as a “domino” effect. PMID:16254054

  18. Ethanol-induced loss of brain cyclic AMP binding proteins: correlation with growth suppression

    SciTech Connect

    Pennington, S.; Kalmus, G.

    1987-05-01

    Brain hypoplasia secondary to maternal ethanol consumption is a common fetal defect observed in all models of fetal alcohol syndrome. The molecular mechanism by which ethanol inhibits growth is unknown but has been hypothesized to involve ethanol-induced changes in the activity of cyclic-AMP stimulated protein kinase. Acute and chronic alcohol exposure elevate cyclic AMP level in many tissues, including brain. This increase in cyclic AMP should increase the phosphorylating activity of kinase by increasing the amount of dissociated (active) kinase catalytic subunit. In 7-day embryonic chick brains, ethanol-induced growth suppression was correlated with increased brain cyclic AMP content but neither basal nor cyclic AMP stimulated kinase catalytic activity was increased. However, the levels of cyclic AMP binding protein (kinase regulatory subunit) were significantly lowered by ethanol exposure. Measured as either /sup 3/H cyclic AMP binding or as 8-azido cyclic AM/sup 32/P labeling, ethanol-exposed brains had significantly less cyclic AMP binding activity (51 +/- 14 versus 29 +/- 10 units/..mu..g protein for 8-azido cyclic AMP binding). These findings suggest that ethanol's effect on kinase activity may involve more than ethanol-induced activation of adenylate cyclase.

  19. Neutrophil activation induced by the lectin KM+ involves binding to CXCR2.

    PubMed

    Pereira-da-Silva, Gabriela; Moreno, Andréa N; Marques, Fabiana; Oliver, Constance; Jamur, Maria Célia; Panunto-Castelo, Ademilson; Roque-Barreira, Maria Cristina

    2006-01-01

    The lectin KM+ from Artocarpus integrifolia, also known as artocarpin, induces neutrophil migration by haptotaxis. The interactions of KM+ with both neutrophils and the extracellular matrix depend on the lectin's ability to recognize mannose-containing glycans. In the present study, we characterized the binding of KM+ to human neutrophils and the responses stimulated by this binding. Exposure to KM+ results in cell polarization, formation of a lamellipodium, and induction of deep ruffles on the cell surface. By fluorescence microscopy, we observed that KM+ is distributed homogeneously over the cell surface. KM+/ligand complexes are rapidly internalized, reaching maximum intracellular concentrations at 120 min, and decreasing thereafter. Furthermore, KM+ binding to the surface of human neutrophils is inhibited by the specific sugars, d-mannose or mannotriose. KM+-induced neutrophil migration is inhibited by pertussis toxin as well as by inhibition of CXCR2 activity. These results suggest that the KM+ ligand on the neutrophil surface is a G protein-coupled receptor (GPCR). The results also suggest that neutrophil migration induced by KM+ involves binding to CXCR2.

  20. Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing

    PubMed Central

    Kisseleva, Natalia; Kraut, Stefanie; Jäschke, Andres; Schiemann, Olav

    2007-01-01

    In ribozyme catalysis, metal ions are generally known to make structural and∕or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper Kd of 0.6±0.2 μM. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be ∼6 Å, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions. PMID:19404418

  1. pSTAT1, pSTAT3, and T-bet as markers of disease activity in chronic inflammatory demyelinating polyradiculoneuropathy.

    PubMed

    Madia, Francesca; Frisullo, Giovanni; Nociti, Viviana; Conte, Amelia; Luigetti, Marco; Del Grande, Alessandra; Patanella, Agata Katia; Iorio, Raffaele; Tonali, Pietro Attilio; Batocchi, Anna Paola; Sabatelli, Mario

    2009-06-01

    Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is considered an auto-immune disorder. We evaluated expression of pSTAT1, T-bet, and pSTAT3 in circulating T-cells, B-cells, and monocytes and spontaneous production of interleukin-17 (IL17), interferon-gamma (IFN gamma), and interleukin-10 (IL10) by peripheral blood mononuclear cells (PBMCs) from 14 active CIDP patients compared with 6 patients with long-lasting remission and 20 controls. Active disease patients showed higher pSTAT1, T-bet, and pSTAT3 in CD4(+) T-cells than controls (p < 0.001, p = 0.0002, p = 0.0097, respectively) and remission patients (p < 0.001, p = 0.0036, p = 0.0008, respectively). pSTAT1, T-bet, and pSTAT3 were also higher in monocytes from active CIDP patients than controls (p = 0.0011, p = 0.0041, p = 0.0413, respectively) and remission patients (p = 0.0073, p = 0.0274, p = 0.0251, respectively). Moreover in CD8(+) T-cells, pSTAT3 expression was higher in active CIDP patients than in remission patients (p = 0.0345) and in controls (p = 0.0023). IL17 and IFN gamma production were significantly higher in active CIDP patients than in controls (p < 0.0395, p = 0.0010, respectively); IFN gamma levels were higher also in remission CIDP patients (p = 0.0073). IL10 levels were higher in active phase patients than in controls (p = 0.0334). Our data suggest that pSTAT1, T-bet, and pSTAT3 can be considered putative markers of disease activity and potential targets for specific therapies.

  2. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation.

    PubMed

    Moncunill-Massaguer, Cristina; Saura-Esteller, José; Pérez-Perarnau, Alba; Palmeri, Claudia Mariela; Núñez-Vázquez, Sonia; Cosialls, Ana M; González-Gironès, Diana M; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-12-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa(-/-)/Bim(-/-) MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway.

  3. The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney.

    PubMed

    O'Brown, Zach K; Van Nostrand, Eric L; Higgins, John P; Kim, Stuart K

    2015-12-01

    Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney.

  4. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses.

    PubMed

    Gay, Gabrielle; Braun, Laurence; Brenier-Pinchart, Marie-Pierre; Vollaire, Julien; Josserand, Véronique; Bertini, Rose-Laurence; Varesano, Aurélie; Touquet, Bastien; De Bock, Pieter-Jan; Coute, Yohann; Tardieux, Isabelle; Bougdour, Alexandre; Hakimi, Mohamed-Ali

    2016-08-22

    An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. PMID:27503074

  5. Accuracy of capillary whole blood international normalized ratio on the CoaguChek S, CoaguChek XS, and i-STAT 1 point-of-care analyzers.

    PubMed

    Karon, Brad S; McBane, Robert D; Chaudhry, Rajeev; Beyer, Lisa K; Santrach, Paula J

    2008-07-01

    We evaluated the accuracy of capillary whole blood international normalized ratio (INR) on the CoaguChek S (Roche Diagnostics, Indianapolis, IN), CoaguChek XS (Roche Diagnostics), and i-STAT 1 (i-STAT, East Windsor, NJ) point-of-care (POC) analyzers compared with venous plasma INRs determined by a reference laboratory method. Overall agreement between POC and laboratory plasma INR was very good, with median bias between capillary whole blood and laboratory plasma INRs varying from 0.0 to -0.2 INR units on all devices. More than 90% of results on the CoaguChek XS and i-STAT 1 and 88% of CoaguChek S results were within 0.4 INR units of the reference laboratory method. The CoaguChek XS and i-STAT 1 demonstrated greater accuracy than the CoaguChek S as measured by the number of results that differed by more than 0.5 INR units from the reference method. Median bias between CoaguChek S capillary whole blood and laboratory plasma INRs changed over time, demonstrating the need for ongoing quality assurance measures for POC INR programs.

  6. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  7. Functional analysis of an auxin-inducible DNA-binding protein gene

    PubMed Central

    Bernstein, Any; Mangeon, Amanda; Almeida-Engler, Janice; Engler, Gilbert; Montagu, Marc Van; Sachetto-Martins, Gilberto; de Oliveira, Dulce Eleonora

    2015-01-01

    Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance. PMID:25482757

  8. Functional analysis of an auxin-inducible DNA-binding protein gene.

    PubMed

    Bernstein, Any; Mangeon, Amanda; Almeida-Engler, Janice; Engler, Gilbert; Van Montagu, Marc; Sachetto-Martins, Gilberto; de Oliveira, Dulce Eleonora

    2015-01-01

    Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin-induced callus formation and reduced apical dominance.

  9. Caerulomycin A Enhances Transforming Growth Factor-β (TGF-β)-Smad3 Protein Signaling by Suppressing Interferon-γ (IFN-γ)-Signal Transducer and Activator of Transcription 1 (STAT1) Protein Signaling to Expand Regulatory T Cells (Tregs)*

    PubMed Central

    Gurram, Rama Krishna; Kujur, Weshely; Maurya, Sudeep K.; Agrewala, Javed N.

    2014-01-01

    Cytokines play a very important role in the regulation of immune homeostasis. Regulatory T cells (Tregs) responsible for the generation of peripheral tolerance are under the tight regulation of the cytokine milieu. In this study, we report a novel role of a bipyridyl compound, Caerulomycin A (CaeA), in inducing the generation of Tregs. It was observed that CaeA substantially up-regulated the pool of Tregs, as evidenced by an increased frequency of CD4+ Foxp3+ cells. In addition, CaeA significantly suppressed the number of Th1 and Th17 cells, as supported by a decreased percentage of CD4+/IFN-γ+ and CD4+/IL-17+ cells, respectively. Furthermore, we established the mechanism and observed that CaeA interfered with IFN-γ-induced STAT1 signaling by augmenting SOCS1 expression. An increase in the TGF-β-mediated Smad3 activity was also noted. Furthermore, CaeA rescued Tregs from IFN-γ-induced inhibition. These results were corroborated by blocking Smad3 activity, which abolished the CaeA-facilitated generation of Tregs. In essence, our results indicate a novel role of CaeA in inducing the generation of Tregs. This finding suggests that CaeA has enough potential to be considered as a potent future drug for the treatment of autoimmunity. PMID:24811173

  10. Carboxy-Terminus Recruitment Induced by Substrate Binding in Eukaryotic Fructose Bis-phosphate Aldolases

    SciTech Connect

    Lafrance-Vanasse,J.; Sygusch, J.

    2007-01-01

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 {angstrom} resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P{sub 1}-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P{sub 1}-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P{sub 1}-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.

  11. Glutamate-induced octamer DNA binding and transcriptional control in cultured radial glia cells.

    PubMed

    López-Bayghen, Esther; Cruz-Solís, Irma; Corona, Matilde; López-Colomé, Ana María; Ortega, Arturo

    2006-08-01

    Glutamate, the main excitatory neurotransmitter in the vertebrate brain, is critically involved in gene expression regulation in neurons and in glia cells. Neuron-glia interactions provide the framework for synaptic plasticity. Retinal and cerebellar radial glia cells surround glutamatergic excitatory synapses and sense synaptic activity through glutamate receptors expressed in their membranes. Several glutamate-dependent membrane to nuclei signaling cascades have been described in these cells. Octamer DNA binding factors, namely Oct-1 and Oct-2 recognize similar DNA sequences on regulatory regions, but their final transcriptional effect depends on several factors. By these means, different responses can be achieved in different cell types. Here, we describe a comparison between the glutamate-induced DNA binding of octamer factors and their functional activities in two important types of radial glia, retinal Müller and cerebellar Bergmann glial cells. While Oct-1 is expressed in both cell types and in both glutamate treatments results in an increase in Oct-1 DNA binding, this complex is capable of transactivating a reporter gene only in Müller glia cells. In contrast, Oct-2 expression is restricted to Bergmann glia cells in which glutamate treatment results in an augmentation of Oct-2 DNA binding complexes and the repression of kainate binding protein gene transcription. Our present findings demonstrate a differential role for Oct-1 and Oct-2 transcription factors in glial glutamate signaling, and further strengthen the notion of an important role for glial cells in glutamatergic transactions in the central nervous system.

  12. Location of inhibitor binding sites in the human inducible prostaglandin E synthase, MPGES1.

    PubMed

    Prage, Edward B; Pawelzik, Sven-Christian; Busenlehner, Laura S; Kim, Kwangho; Morgenstern, Ralf; Jakobsson, Per-Johan; Armstrong, Richard N

    2011-09-01

    The inducible microsomal prostaglandin E(2) synthase 1 (MPGES1) is an integral membrane protein coexpressed with and functionally coupled to cyclooxygenase 2 (COX-2) generating the pro-inflammatory molecule PGE(2). The development of effective inhibitors of MPGES1 holds promise as a highly selective route for controlling inflammation. In this paper, we describe the use of backbone amide H/D exchange mass spectrometry to map the binding sites of different types of inhibitors of MPGES1. The results reveal the locations of specific inhibitor binding sites that include the GSH binding site and a hydrophobic cleft in the protein thought to accommodate the prostaglandin H(2) substrate. In the absence of three-dimensional crystal structures of the enzyme-bound inhibitors, the results provide clear physical evidence that three pharmacologically active inhibitors bind in a hydrophobic cleft composed of sections of transmembrane helices Ia, IIb, IIIb, and IVb at the interface of subunits in the trimer. In principle, the H/D exchange behavior of the protein can be used as a preliminary guide for optimization of inhibitor efficacy. Finally, a comparison of the structures and H/D exchange behavior of MPGES1 and the related enzyme MGST1 in the presence of glutathione and the inhibitor glutathione sulfonate confirms the unusual observation that two proteins from the same superfamily harbor GSH binding sites in different locations.

  13. Laser-Induced Fluorescence Analysis of Protein-Based Binding Media

    NASA Astrophysics Data System (ADS)

    Nevin, A.; Cather, S.; Anglos, D.; Fotakis, Costas

    Laser-induced fluorescence of intrinsic fluorophores of organic media found in paintings (casein, animal glue and egg proteins) provides a means of characterising general classes of media depending on the amino acid composition and presence of degradation cross-linkages. Wavelength dependence of spectra is investigated for non-destructive and non-invasive analyses of thin films of protein-based binding media.

  14. Induced Long-Range Attractive Potentials of Human Serum Albumin by Ligand Binding

    SciTech Connect

    Sato, Takaaki; Komatsu, Teruyuki; Nakagawa, Akito; Tsuchida, Eishun

    2007-05-18

    Small-angle x-ray scattering and dielectric spectroscopy investigation on the solutions of recombinant human serum albumin and its heme hybrid revealed that heme incorporation induces a specific long-range attractive potential between protein molecules. This is evidenced by the enhanced forward intensity upon heme binding, despite no hindrance to rotatory Brownian motion, unbiased colloid osmotic pressure, and discontiguous nearest-neighbor distance, confirming monodispersity of the proteins. The heme-induced potential may play a trigger role in recognition of the ligand-filled human serum albumins in the circulatory system.

  15. Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer.

    PubMed

    Daniel, Kenyon G; Chen, Di; Yan, Bing; Dou, Q Ping

    2007-01-01

    The trace element copper is vital to the healthy functioning of organisms. Copper is used in a multitude of cellular activities including respiration, angiogenesis, and immune responses. Recently, copper has become a focus in medical research ranging from Alzheimer's disease to cancer. Copper modulation has been suggested to be a potential modality for therapy in these diseases. Several copper-binding compounds have been found to spontaneously complex with copper and form active proteasome inhibitors and apoptosis inducers. This review examines compounds in the quinoline and dithiocarbamate families and from the National Cancer Institute (NCI) Diversity Set that bind with copper and act as anticancer agents. In each case, it is shown that these compounds can bind with copper, inhibit the proteasome activity, and induce apoptosis in cancer cells. These activities are absent when copper is not present. Compounds alone, clioquinol and pyrrolidinedithiocarbamate as examples, are shown to have no effects in normal breast cells. Current research suggests that a possible therapeutic modality for cancer may be developed using the difference of high copper load in tumors versus low copper load in normal cells. This strategy would convert tumor cellular copper into a potent, specific proteasome inhibitor and apoptosis inducer. Thus, this approach could pave the way for the development of nontoxic anticancer therapy.

  16. PUMA promotes Bax translocation by competitive binding to Bcl-Xl during UV-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Zhang, Yingjie; Xing, Da; Wu, Yinyuan; Liu, Lei

    2008-02-01

    Ultraviolet (UV) irradiation can induce apoptosis through both the membrane death receptor and the intrinsic apoptotic signaling pathways as DNA-damaging agents. PUMA, a BH3-only Bcl-2 family protein, plays an essential role in DNA damage-induced apoptosis. Bax, also a Bcl-2 family member, translocates from the cytosol to the mitochondrial membrane during UV-induced apoptosis. However, the regulation of Bax activation induced by UV irradiation remains poorly understood. In this study, the FRET (fluorescence resonance energy transfer) technique was used to study the interactions of Bax, Bcl-Xl, and PUMA in ASTC-a-1 cells. The results show that Bax translocated from the cytosol to the mitochondrial membrane at about 7 h after UV irradiation, and the translocation can not be blocked completely when overexpressed Bcl-xl. Moreover, The interaction of Bax and Bcl-Xl weakened markedly. In addition, Co-immunoprecipitation shows that PUMA released Bax by directly binding to Bcl-XL after UV irradiation in ASTC-a-1 cells. Taken together, these results indicated that PUMA can promote Bax translocation by binding to Bcl-Xl during UV-induced apoptosis.

  17. [Conformational changes of actin induced by strong or weak myosin subfragment-1 binding].

    PubMed

    Dedova, I V; Avrova, S V; Vikhoreva, N N; Vikhorev, R G; Hazlett, T L; Van der Meer, W; Dos Remedios, C G; Borovikov, Iu S

    2004-01-01

    Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

  18. Organization of the Mouse RNA-specific Adenosine Deaminase Adar1 Gene 5’-Region and Demonstration of STAT1-independent, STAT2-dependent Transcriptional Activation by Interferon

    PubMed Central

    George, Cyril X.; Das, Sonali; Samuel, Charles E.

    2008-01-01

    The p150 form of the RNA-specific adenosine deaminase ADAR1 is interferon-inducible and catalyzes A-to-I editing of viral and cellular RNAs. We have characterized mouse genomic clones containing the promoter regions required for Adar1 gene transcription and analyzed interferon induction of the p150 protein using mutant mouse cell lines. Transient transfection analyses using reporter constructs led to the identification of three promoters, one interferon-inducible (PA) and two constitutively active (PB and PC). The TATA-less PA promoter, characterized by the presence of a consensus ISRE element and a PKR kinase KCS-like element, directed interferon-inducible reporter expression in rodent and human cells. Interferon induction of p150 was impaired in mouse cells deficient in IFNAR receptor, JAK1 kinase or STAT2 but not STAT1. Whereas Adar1 gene organization involving multiple promoters and alternative exon 1 structures was highly preserved, sequences of the promoters and exon 1 structures were not well conserved between human and mouse. PMID:18774582

  19. Binding characteristics of radioiodinated crystal - induced chemotactic factor to human neutrophils

    SciTech Connect

    Spilberg, I.; Mehta, J.

    1984-12-01

    The binding of radiolabeled crystal-induced chemotactic factor (CCF) to human neutrophils is characterized. Binding of /sup 125/I-CCF to the cells was higher at 4/sup 0/C than at 24/sup 0/ or 37/sup 0/C and was found to be independent of Ca/sup 2 +/ and Mg/sup 2 +/ ion concentration. The binding showed a pH optimum of 6.0. Tosylamido phenylethyl chloromethyl ketone at 100 ..mu..mol/L concentration inhibited 20% of /sup 125/I-CCF binding, but phenylmethylsulfonyl fluoride at 200 ..mu..mol/L had no effect. Approximately 50% of the cell-associated /sup 125/I-CCF was released after treatment with proteases. The nonspecific uptake by the cells, as measured by the uptake of /sup 3/H-sucrose and /sup 14/C-inulin in the presence of CCF, was negligible. After the steady-state binding of /sup 125/I-CCF to the cells, approx. 15% of the cell-associated radioactivity at 4/sup 0/C and 40% to 50% at 24/sup 0/ and 37/sup 0/C was released into the medium after 60 minutes of incubation in medium alone. Dissociation of the radioactive material was not affected by the presence of tosylamido phenylethyl chloromethyl ketone or phenanthroline in the media. The dissociated material was determined to be degraded /sup 125/I-CCF, suggesting that degradation of /sup 125/I-CCF occurs after binding to its specific receptor on the human neutrophil. 22 references, 3 figures, 2 tables.

  20. Binding Mode and Induced Fit Predictions for Prospective Computational Drug Design.

    PubMed

    Grebner, Christoph; Iegre, Jessica; Ulander, Johan; Edman, Karl; Hogner, Anders; Tyrchan, Christian

    2016-04-25

    Computer-aided drug design plays an important role in medicinal chemistry to obtain insights into molecular mechanisms and to prioritize design strategies. Although significant improvement has been made in structure based design, it still remains a key challenge to accurately model and predict induced fit mechanisms. Most of the current available techniques either do not provide sufficient protein conformational sampling or are too computationally demanding to fit an industrial setting. The current study presents a systematic and exhaustive investigation of predicting binding modes for a range of systems using PELE (Protein Energy Landscape Exploration), an efficient and fast protein-ligand sampling algorithm. The systems analyzed (cytochrome P, kinase, protease, and nuclear hormone receptor) exhibit different complexities of ligand induced fit mechanisms and protein dynamics. The results are compared with results from classical molecular dynamics simulations and (induced fit) docking. This study shows that ligand induced side chain rearrangements and smaller to medium backbone movements are captured well in PELE. Large secondary structure rearrangements, however, remain challenging for all employed techniques. Relevant binding modes (ligand heavy atom RMSD < 1.0 Å) can be obtained by the PELE method within a few hours of simulation, positioning PELE as a tool applicable for rapid drug design cycles. PMID:26974351

  1. Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

    PubMed Central

    Han, Song; Wang, Guiyun; Qi, Xiang; Englander, Ella W.; Greeley, George H.

    2008-01-01

    Apelin is the endogenous ligand for the APJ receptor; both are expressed in the gastrointestinal tract. Experimental colitis in rodents and inflammatory bowel disease in humans are associated with increased intestinal apelin production. Our aim was to use LPS and proinflammatory cytokine-treated (IL-6 and IFN-γ) rodents or enteric cells to identify signaling mechanisms underlying inflammation-induced enteric apelin expression. LPS, IL-6, or IFN-γ treatment of rodents increased enteric apelin expression. Pharmacological blockade of Jak/Stat signaling or IL-6 antibody administration inhibited elevations in enteric apelin expression. Transient transfection experiments showed that LPS, IL-6, or IFN-γ increased apelin expression by stimulation of apelin promoter activity, and blockade of Jak/Stat signaling abolished elevations in apelin promoter activity. A chromatin immunoprecipitation assay showed that IL-6 induced binding of phospho-Stat3 to a putative Stat3 site in the apelin promoter; mutation of this site abrogated the LPS-induced elevation in apelin promoter activity. Together, our findings indicate that binding of phospho-Stat3 to the apelin promoter is the final step underlying proinflammatory cytokine-induced enteric apelin expression during intestinal inflammation. PMID:18818315

  2. A zinc-binding site by negative selection induces metallodrug susceptibility in an essential chaperonin.

    PubMed

    Cun, Shujian; Sun, Hongzhe

    2010-03-16

    GroES is an indispensable chaperonin virtually found throughout all life forms. Consequently, mutations of this protein must be critically scrutinized by natural selection. Nevertheless, the homolog from a potentially virulent gastric pathogen, Helicobacter pylori, strikingly features a histidine/cysteine-rich C terminus that shares no significant homology with other family members. Additionally, three more (H45, C51, and C53) are uniquely present in its apical domain. The statistical analyses show that these residues may have originated from negative selection, presumably driven by either dependent or independent amino acid mutations. In the absence of the C-terminal metal-binding domain, the mutant protein still exhibits a substantial capacity for zinc binding in vivo. The biochemical properties of site-directed mutants indicate that H45, C51, and C53 make up an oxidation-sensitive zinc-binding site that may donate the bound metal to a zinc acceptor. Of interest, bismuth antiulcer drugs strongly bind at this site (K(d) of approximately 7 x 10(-26) M), replacing the bound zinc and consequently inducing the disruption of the quaternary structure. Because biological features by negative selection are usually inert to change during evolution, this study sheds light on a promising field whereby medicines can be designed or improved to specifically target the residues that uniquely evolved in pathogenic proteins so as to retard the emergence of drug resistance. PMID:20194796

  3. Thermodynamic basis for the optimization of binding-induced biomolecular switches and structure-switching biosensors.

    PubMed

    Vallée-Bélisle, Alexis; Ricci, Francesco; Plaxco, Kevin W

    2009-08-18

    Binding-induced biomolecular switches are used throughout nature and, increasingly, throughout biotechnology for the detection of chemical moieties and the subsequent transduction of this detection into useful outputs. Here we show that the thermodynamics of these switches are quantitatively described by a simple 3-state population-shift model, in which the equilibrium between a nonbinding, nonsignaling state and the binding-competent, signaling state is shifted toward the latter upon target binding. Because of this, their performance is determined by the tradeoff inherent to their switching thermodynamics; while a switching equilibrium constant favoring the nonbinding, nonsignaling, conformation ensures a larger signal change (more molecules are poised to respond), it also reduces affinity (binding must overcome a more unfavorable conformational free energy). We then derive and employ the relationship between switching thermodynamics and switch signaling to rationally tune the dynamic range and detection limit of a representative structure-switching biosensor, a molecular beacon, over 4 orders of magnitude. These findings demonstrate that the performance of biomolecular switches can be rationally tuned via mutations that alter their switching thermodynamics and suggest a mechanism by which the performance of naturally occurring switches may have evolved.

  4. Binding-Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids.

    PubMed

    Zhang, Hongquan; Lai, Maode; Zuehlke, Albert; Peng, Hanyong; Li, Xing-Fang; Le, X Chris

    2015-11-23

    We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly.

  5. Aptamer/target binding-induced triple helix forming for signal-on electrochemical biosensing.

    PubMed

    Mao, Yinfei; Liu, Jinquan; He, Dinggen; He, Xiaoxiao; Wang, Kemin; Shi, Hui; Wen, Li

    2015-10-01

    Owing to its diversified structures, high affinity, and specificity for binding a wide range of non-nucleic acid targets, aptamer is a useful molecular recognition tool for the design of various biosensors. Herein, we report a new signal-on electrochemical biosensing platform which is based on an aptamer/target binding-induced strand displacement and triple-helix forming. The biosensing platform is composed of a signal transduction probe (STP) modified with a methylene blue (MB) and a sulfhydryl group, a triplex-forming oligonucleotides probe (TFO) and a target specific aptamer probe (Apt). Through hybridization with the TFO probe and the Apt probe, the self-assembled STP on Au electrode via Au-S bonding keeps its rigid structure. The MB on the STP is distal to the Au electrode surface. It is eT off state. Target binding releases the Apt probe and liberates the end of the MB tagged STP to fold back and form a triplex-helix structure with TFO (STP/TFO/STP), allowing MB to approach the Au electrode surface and generating measurable electrochemical signals (eT ON). As test for the feasibility and universality of this signal-on electrochemical biosensing platform, two aptamers which bind to adenosine triphosphate (ATP) and human α-thrombin (Tmb), respectively, are selected as models. The detection limit of ATP was 7.2 nM, whereas the detection limit of Tmb was 0.86 nM.

  6. DNA-binding activity of TNF-{alpha} inducing protein from Helicobacter pylori

    SciTech Connect

    Kuzuhara, T. Suganuma, M.; Oka, K.; Fujiki, H.

    2007-11-03

    Tumor necrosis factor-{alpha} (TNF-{alpha}) inducing protein (Tip{alpha}) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-{alpha} and chemokine genes and activation of nuclear factor-{kappa}B. Since Tip{alpha} enters gastric cancer cells, the Tip{alpha} binding molecules in the cells should be investigated. The direct DNA-binding activity of Tip{alpha} was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tip{alpha} and DNA, revealed that the affinity of Tip{alpha} for (dGdC)10 is 2400 times stronger than that of del-Tip{alpha}, an inactive Tip{alpha}. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tip{alpha}. And the DNA-binding activity of Tip{alpha} was first demonstrated with a molecule secreted from H. pylori.

  7. Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

    PubMed Central

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

  8. DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

    PubMed

    Tse, Margaret J; Chu, Brian K; Roy, Mahua; Read, Elizabeth L

    2015-10-20

    Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

  9. Identification of an Aptamer Binding to Human Osteogenic-Induced Progenitor Cells

    PubMed Central

    Niederlaender, Jan; Aicher, Wilhelm K.; Reinert, Siegmar; Schweizer, Ernst; Wendel, Hans-Peter; Alexander, Dorothea

    2013-01-01

    The aim of this study was to generate a specific aptamer against human jaw periosteal cells (JPCs) for tissue engineering applications in oral and maxillofacial surgery. This aptamer should serve as a capture molecule to enrich or even purify osteogenic progenitor cells from JPCs or from adult stem cells of other sources. Using systematic evolution of ligands by exponential enrichment (SELEX), we generated the first aptamer to specifically bind to human osteogenically induced JPCs. We did not detect any binding of the aptamer to undifferentiated JPCs, adipogenically and chondrogenically induced JPCs, or to any other cell line tested. However, similar binding patterns of the identified aptamer 74 were detected with mesenchymal stromal cells (MSCs) derived from placental tissue and bone marrow. After cell sorting, we analyzed the expression of osteogenic marker genes in the aptamer 74-positive and aptamer 74-negative fractions and detected no significant differences. Additionally, the analysis of the mineralization capacity revealed a slight tendency for the aptamer positive fraction to have a higher osteogenic potential. In terms of proliferation, JPCs growing in aptamer-coated wells showed increased proliferation rates compared with the controls. Herein, we report the development of an innovative approach for tissue engineering applications. Further studies should be conducted to modify and improve the specificity of the generated aptamer. PMID:23289534

  10. Adenovirus carrying gene encoding Haliotis discus discus sialic acid binding lectin induces cancer cell apoptosis.

    PubMed

    Yang, Xinyan; Wu, Liqin; Duan, Xuemei; Cui, Lianzhen; Luo, Jingjing; Li, Gongchu

    2014-06-30

    Lectins exist widely in marine bioresources such as bacteria, algae, invertebrate animals and fishes. Some purified marine lectins have been found to elicit cytotoxicity to cancer cells. However, there are few reports describing the cytotoxic effect of marine lectins on cancer cells through virus-mediated gene delivery. We show here that a replication-deficient adenovirus-carrying gene encoding Haliotis discus discus sialic acid binding lectin (Ad.FLAG-HddSBL) suppressed cancer cell proliferation by inducing apoptosis, as compared to the control virus Ad.FLAG. A down-regulated level of anti-apoptosis factor Bcl-2 was suggested to be responsible for the apoptosis induced by Ad.FLAG-HddSBL infection. Further subcellular localization studies revealed that HddSBL distributed in cell membrane, ER, and the nucleus, but not in mitochondria and Golgi apparatus. In contrast, a previously reported mannose-binding lectin Pinellia pedatisecta agglutinin entered the nucleus as well, but did not distribute in inner membrane systems, suggesting differed intracellular sialylation and mannosylation, which may provide different targets for lectin binding. Further cancer-specific controlling of HddSBL expression and animal studies may help to provide insights into a novel way of anti-cancer marine lectin gene therapy. Lectins may provide a reservoir of anti-cancer genes.

  11. A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution.

    PubMed

    Galfrè, Elena; Galeno, Lauretta; Moran, Oscar

    2012-11-01

    Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs-intracellular loop interaction in a way that would facilitate the open state of the channel.

  12. Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

    PubMed Central

    Theodosiou, Marina; Widmaier, Moritz; Böttcher, Ralph T; Rognoni, Emanuel; Veelders, Maik; Bharadwaj, Mitasha; Lambacher, Armin; Austen, Katharina; Müller, Daniel J; Zent, Roy; Fässler, Reinhard

    2016-01-01

    Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner. DOI: http://dx.doi.org/10.7554/eLife.10130.001 PMID:26821125

  13. Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression.

    PubMed

    Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X; Walterscheid, Jeffrey P; Ullrich, Stephen E

    2004-03-15

    Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependent manner. The release of biological response modifiers, particularly prostaglandin E2 (PGE2), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE2 secretion. Jet fuel-induced PGE2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin. PMID:15020195

  14. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

    PubMed

    Kim, Bae-Hoon; Chee, Jonathan D; Bradfield, Clinton J; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D

    2016-05-01

    Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases. PMID:27092805

  15. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

    PubMed

    Kim, Bae-Hoon; Chee, Jonathan D; Bradfield, Clinton J; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D

    2016-05-01

    Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.

  16. IFN-induced Guanylate Binding Proteins in Inflammasome Activation and Host Defense

    PubMed Central

    Kim, Bae-Hoon; Chee, Jonathan D.; Bradfield, Clinton J.; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D.

    2016-01-01

    Traditional views of the inflammasome highlight pre-existing core components being assembled under basal conditions shortly after infection or tissue damage. Recent work, however, suggests the inflammasome machinery is also subject to tunable or inducible signals that may accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these immune signals operate downstream of interferon (IFN) receptors to elicit inflammasome regulators, including a new family of IFN-induced GTPases termed guanylate binding proteins (GBPs). Here, we examine the critical roles for IFN-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity against a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential impact of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases. PMID:27092805

  17. CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells.

    PubMed

    Gireesh, K K; Rashid, Aijaz; Chakraborti, Soumyananda; Panda, Dulal; Manna, Tapas

    2012-09-01

    A plant dictamine analog, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ~0.4 μM. Isothermal titration calorimetry revealed that CIL-102-tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (ΔC(p) = -790 cal mol(-1) K(-1)) with an enthalpy-entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ~1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule-kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53-p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine. PMID:22705644

  18. Cold inducible RNA binding protein upregulation in pituitary corticotroph adenoma induces corticotroph cell proliferation via Erk signaling pathway

    PubMed Central

    Fu, Wei; Tang, Hao; Chen, Xiao; Zhao, Yao; Zheng, Lili; Pan, Sijian; Wang, Weiqing; Bian, Liuguan; Sun, Qingfang

    2016-01-01

    Cushing's disease is caused by pituitary corticotroph adenoma, and the pathogenesis of it has remained obscure. Here, we showed that cold inducible RNA binding protein (CIRP) was markedly elevated in corticotroph tumors. Forced overexpression of CIRP in murine AtT20 pituitary corticotroph cell line increased corticotroph precursor hormone proopiomelanocortin (POMC) transcription, ACTH secretion and cellular proliferation. In vivo, CIRP overexpression promotes murine corticotroph tumor growth and enhances ACTH production. Mechanistically, we show that CIRP could promote AtT20 cells proliferation by inducing cyclinD1 and decreasing p27 expression via Erk1/2 signaling pathway. Clinically, CIRP overexpression is significantly correlated with Cushing's disease recurrence. CIRP appears to play a critical tumorigenesis function in Cushing's disease and its expression might be a useful biomarker for tumor recurrence. PMID:26824322

  19. Impact of mannose-binding lectin deficiency on radiocontrast-induced renal dysfunction.

    PubMed

    Osthoff, Michael; Trendelenburg, Marten

    2013-01-01

    Contrast-induced nephropathy (CIN) is the third leading cause of acute renal failure in hospitalized patients. Endothelial dysfunction, renal medullary ischemia, and tubular toxicity are regarded as the most important factors in the pathogenesis of CIN. Mannose-binding lectin (MBL), a pattern recognition protein of the lectin pathway of complement, has been found to aggravate and mediate tissue damage during experimental renal ischemia/reperfusion (I/R) injury which was alleviated by inhibition with C1 inhibitor, a potent MBL, and lectin pathway inhibitor. In this paper, we highlight the potential role of MBL in the pathogenesis of human CIN. In experimental I/R models, MBL was previously found to induce tubular cell death independent of the complement system. In addition, after binding to vascular endothelial cells, MBL and its associated serine proteases were able to trigger a proinflammatory reaction and contribute to endothelial dysfunction. In humans, urinary MBL was increased after administration of contrast media and in individuals with CIN. Moreover, individuals with normal/high MBL levels were at increased risk to develop radiocontrast-induced renal dysfunction. Hence, MBL and the lectin pathway seem to be a promising target given that a licensed, powerful, human recombinant inhibitor exits to be added to the scarce armamentarium currently available for prophylaxis of CIN.

  20. Impact of Mannose-Binding Lectin Deficiency on Radiocontrast-Induced Renal Dysfunction

    PubMed Central

    Osthoff, Michael; Trendelenburg, Marten

    2013-01-01

    Contrast-induced nephropathy (CIN) is the third leading cause of acute renal failure in hospitalized patients. Endothelial dysfunction, renal medullary ischemia, and tubular toxicity are regarded as the most important factors in the pathogenesis of CIN. Mannose-binding lectin (MBL), a pattern recognition protein of the lectin pathway of complement, has been found to aggravate and mediate tissue damage during experimental renal ischemia/reperfusion (I/R) injury which was alleviated by inhibition with C1 inhibitor, a potent MBL, and lectin pathway inhibitor. In this paper, we highlight the potential role of MBL in the pathogenesis of human CIN. In experimental I/R models, MBL was previously found to induce tubular cell death independent of the complement system. In addition, after binding to vascular endothelial cells, MBL and its associated serine proteases were able to trigger a proinflammatory reaction and contribute to endothelial dysfunction. In humans, urinary MBL was increased after administration of contrast media and in individuals with CIN. Moreover, individuals with normal/high MBL levels were at increased risk to develop radiocontrast-induced renal dysfunction. Hence, MBL and the lectin pathway seem to be a promising target given that a licensed, powerful, human recombinant inhibitor exits to be added to the scarce armamentarium currently available for prophylaxis of CIN. PMID:24386641

  1. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms.

  2. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms. PMID:27063510

  3. Induced circular dichroism as a tool to investigate the binding of drugs to carrier proteins: Classic approaches and new trends.

    PubMed

    Tedesco, Daniele; Bertucci, Carlo

    2015-09-10

    Induced circular dichroism (ICD) is a spectroscopic phenomenon that provides versatile and useful methods for characterizing the structural and dynamic properties of the binding of drugs to target proteins. The understanding of biorecognition processes at the molecular level is essential to discover and validate new pharmacological targets, and to design and develop new potent and selective drugs. The present article reviews the main applications of ICD to drug binding studies on serum carrier proteins, going from the classic approaches for the derivation of drug binding parameters and the identification of binding sites, to an overview of the emerging trends for the characterization of binding modes by means of quantum chemical (QC) techniques. The advantages and limits of the ICD methods for the determination of binding parameters are critically reviewed; the capability to investigate the binding interactions of drugs and metabolites to their target proteins is also underlined, as well as the possibility of characterizing the binding sites to obtain a complete picture of the binding mechanism and dynamics. The new applications of ICD methods to identify stereoselective binding modes of drug/protein complexes are then reviewed with relevant examples. The combined application of experimental ICD spectroscopy and QC calculations is shown to identify qualitatively the bound conformations of ligands to target proteins even in the absence of a detailed structure of the binding sites, either obtained from experimental X-ray crystallography and NMR measurements or from computational models of the complex.

  4. Fasting induces the generation of serum thyronine-binding globulin in Zucker rats

    SciTech Connect

    Young, R.A.; Rajatanavin, R.; Moring, A.F.; Braverman, L.E.

    1985-04-01

    Five-month-old lean and obese Zucker rats were fasted for up to 7 days (lean rats) or 28 days (obese rats), and serum total and free T4 and T3 concentrations, percent free T4 and T3 by equilibrium dialysis, and the binding of (/sup 125/I) T4 to serum proteins by gel electrophoresis were measured. In the lean rats, a 4- or 7-day fast resulted in significant decreases in serum total and free T4 and T3 concentrations. There was a decrease in the percent free T3 after 7 days of starvation. In contrast, a 4- or 7-day fast did not alter any of these variables in the obese rats. However, after 14 or more days of starvation, serum total T4 and T3 concentrations increased, and the percent free T4 and T3 decreased, resulting in no change in the serum free T4 or T3 concentrations in the obese rats. The percent of (/sup 125/I)T4 bound to serum thyronine-binding globulin increased and the percent bound to thyronine-binding prealbumin decreased with the duration of the fast in both the lean and obese rats. The increase in serum thyronine-binding globulin binding of T4 can explain the increase in serum total T4 and T3 concentrations, the decrease in percent free T4 and T3, and the normal free hormone concentration in the long term fasted obese rats. The findings in the lean rats appear to be due to a combination of the known central hypothyroidism that occurs during 4-7 days of fasting and the fasting-induced changes in T4 binding in serum. Changes in T4 and T3 binding in serum during fasting in the rat must be considered when the effects of fasting on serum concentrations of the thyroid hormones, thyroid hormone kinetics, and the peripheral action of the thyroid hormones are evaluated.

  5. Confinement induced binding in noble gas atoms within a BN-doped carbon nanotube

    NASA Astrophysics Data System (ADS)

    Chakraborty, Debdutta; Chattaraj, Pratim Kumar

    2015-02-01

    Confinement induced binding interaction patterns for noble gas atoms (Hen/m, Arn, Krn; n = 2, m = 3) atoms inside pristine and -BN doped (3, 3) single walled carbon nanotube (SWCNT) have been studied through density functional theory calculations. The kinetic stability for He dimer and trimer has been investigated at 100 K and 300 K through an ab initio molecular dynamics simulation. The positive role of doping in SWCNT in enhancing the nature of interaction as well as the kinetic stability of the said systems has been found.

  6. Identification and characterization of an antigen binding receptor on an inducible suppressor T cell hybridoma specific for GAT

    SciTech Connect

    Sorensen, C.M.; Pierce, C.W.

    1986-03-05

    C57BL/10 spleen cells were incubated for 18 hrs with monoclonal GAT-TsF1 and soluble GAT, and fused with BW5147. Hybridomas that did not produce GAT-TsF2 constitutively but could be induced by GAT-TsF1 to do so were cloned. One such clone (1556A2.1) was shown to bind GAT via a cell surface structure having the properties of a classical receptor. Binding of /sup 125/I-GAT was saturable and maximal by 4 hrs at 4/sup 0/C. Scatchard analysis indicated 5.8 x 10/sup 4/ binding sites per cell with an avidity of 8 x 10/sup -14/M. Binding of /sup 125/I-GAT was specific because cold GAT, GT, and GLT competed for binding, whereas BSA and GA did not. Furthermore, monoclonal antibody against a shared anti-GAT idiotype also blocked binding of /sup 125/I-GAT. Finally, the antigen-binding receptor isolated from uninduced 1556A2.1 hybridoma failed to complement independently isolated I-J/sup +/ chain from TsF2 obtained from induced 1556A2.1. These data suggest the presence of a molecule on the Ts/sub 2/ cell capable of binding antigen which is related to, but functionally different from, the TsF2 antigen-binding chain.

  7. Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death

    PubMed Central

    Bitomsky, Nadja; Conrad, Elisa; Moritz, Christian; Polonio-Vallon, Tilman; Sombroek, Dirk; Schultheiss, Kathrin; Glas, Carolina; Greiner, Vera; Herbel, Christoph; Mantovani, Fiamma; del Sal, Giannino; Peri, Francesca; Hofmann, Thomas G.

    2013-01-01

    Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1–HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis. PMID:24145406

  8. Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome.

    PubMed

    Yang, Weng-Lang; Sharma, Archna; Wang, Zhimin; Li, Zhigang; Fan, Jie; Wang, Ping

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern (DAMP) molecule which stimulates proinflammatory cytokine release in hemorrhage and sepsis. Under these medical conditions, disruption of endothelial homeostasis and barrier integrity, typically induced by proinflammatory cytokines, is an important factor contributing to morbidity and mortality. However, the role of CIRP in causing endothelial dysfunction has not been investigated. In this study, we show that intravenous injection of recombinant murine CIRP (rmCIRP) in C57BL/6 mice causes lung injury, evidenced by vascular leakage, edema, increased leukocyte infiltration and cytokine production in the lung tissue. The CIRP-induced lung damage is accompanied with endothelial cell (EC) activation marked by upregulation of cell-surface adhesion molecules E-selectin and ICAM-1. Using in vitro primary mouse lung vascular ECs (MLVECs), we demonstrate that rmCIRP treatment directly increases the ICAM-1 protein expression and activates NAD(P)H oxidase in MLVECs. Importantly, CIRP stimulates the assembly and activation of Nlrp3 inflammasome in MLVECs accompanied with caspase-1 activation, IL-1β release and induction of proinflammatory cell death pyroptosis. Finally, our study demonstrates CIRP-induced EC pyroptosis in the lungs of C57BL/6 mice for the first time. Taken together, the released CIRP in shock can directly activate ECs and induce EC pyroptosis to cause lung injury. PMID:27217302

  9. A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation

    PubMed Central

    Moncunill-Massaguer, Cristina; Saura-Esteller, José; Pérez-Perarnau, Alba; Palmeri, Claudia Mariela; Núñez-Vázquez, Sonia; Cosialls, Ana M.; González-Gironès, Diana M.; Pomares, Helena; Korwitz, Anne; Preciado, Sara; Albericio, Fernando; Lavilla, Rodolfo; Pons, Gabriel; Langer, Thomas; Iglesias-Serret, Daniel; Gil, Joan

    2015-01-01

    We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa−/−/Bim−/− MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway. PMID:26497683

  10. Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome

    PubMed Central

    Yang, Weng-Lang; Sharma, Archna; Wang, Zhimin; Li, Zhigang; Fan, Jie; Wang, Ping

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern (DAMP) molecule which stimulates proinflammatory cytokine release in hemorrhage and sepsis. Under these medical conditions, disruption of endothelial homeostasis and barrier integrity, typically induced by proinflammatory cytokines, is an important factor contributing to morbidity and mortality. However, the role of CIRP in causing endothelial dysfunction has not been investigated. In this study, we show that intravenous injection of recombinant murine CIRP (rmCIRP) in C57BL/6 mice causes lung injury, evidenced by vascular leakage, edema, increased leukocyte infiltration and cytokine production in the lung tissue. The CIRP-induced lung damage is accompanied with endothelial cell (EC) activation marked by upregulation of cell-surface adhesion molecules E-selectin and ICAM-1. Using in vitro primary mouse lung vascular ECs (MLVECs), we demonstrate that rmCIRP treatment directly increases the ICAM-1 protein expression and activates NAD(P)H oxidase in MLVECs. Importantly, CIRP stimulates the assembly and activation of Nlrp3 inflammasome in MLVECs accompanied with caspase-1 activation, IL-1β release and induction of proinflammatory cell death pyroptosis. Finally, our study demonstrates CIRP-induced EC pyroptosis in the lungs of C57BL/6 mice for the first time. Taken together, the released CIRP in shock can directly activate ECs and induce EC pyroptosis to cause lung injury. PMID:27217302

  11. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    SciTech Connect

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  12. Subchronic treatment with antiepileptic drugs modifies pentylenetetrazol-induced seizures in mice: Its correlation with benzodiazepine receptor binding

    PubMed Central

    Rocha, Luisa

    2008-01-01

    Experiments using male CD1 mice were carried out to investigate the effects of subchronic (daily administration for 8 days) pretreatments with drugs enhancing GABAergic transmission (diazepam, 10 mg/kg, ip; gabapentin, 100 mg/kg, po; or vigabatrin, 500 mg/kg, po) on pentylenetetrazol (PTZ)-induced seizures, 24 h after the last injection. Subchronic administration of diazepam reduced latencies to clonus, tonic extension and death induced by PTZ. Subchronic vigabatrin produced enhanced latency to the first clonus but faster occurrence of tonic extension and death induced by PTZ. Subchronic gabapentin did not modify PTZ-induced seizures. Autoradiography experiments revealed reduced benzodiazepine receptor binding in several brain areas after subchronic treatment with diazepam or gabapentin, whereas subchronic vigabatrin did not induce significant receptor changes. The present results indicate differential effects induced by the subchronic administration of diazepam, vigabatrin, and gabapentin on the susceptibility to PTZ-induced seizures, benzodiazepine receptor binding, or both. PMID:18830436

  13. SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR

    PubMed Central

    Thomas, J. Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R.; Moos, Malcolm

    2016-01-01

    In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface. PMID:27101391

  14. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation.

    PubMed

    Nauta, Alma J; Trouw, Leendert A; Daha, Mohamed R; Tijsma, Odette; Nieuwland, Rienk; Schwaeble, Wilhelm J; Gingras, Alexandre R; Mantovani, Alberto; Hack, Erik C; Roos, Anja

    2002-06-01

    Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.

  15. Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.

    PubMed

    Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

    2007-05-01

    Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

  16. Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

    PubMed Central

    Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

    2007-01-01

    Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

  17. Hypoxia-inducible factor 2alpha binds to cobalt in vitro.

    PubMed

    Yuan, Y; Beitner-Johnson, D; Millhorn, D E

    2001-11-01

    The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element (HRE). The alpha subunit of the HIF transcription factors is degraded by proteasome pathways during normoxia, but stabilized under hypoxic conditions. It has previously been established that cobalt causes accumulation of HIF-2alpha and HIF-1alpha. However, little is known about the mechanism by which cobalt mimics hypoxia and stabilizes these transcription factors. We show here that cobalt binds directly to HIF-2alpha in vitro with a high affinity and in an oxygen-dependent manner. We found that HIF-2alpha, which had been stabilized with a proteasome inhibitor, could bind to cobalt, whereas hypoxia-stabilized HIF-2alpha could not. Mutations within the oxygen-dependent degradation domain of HIF-2alpha prevented cobalt binding and led to accumulation of HIF-2alpha during normoxia. This suggests that transition metal such as iron may play a role in regulation of HIF-2alpha in vivo. PMID:11688986

  18. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    PubMed

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients. PMID:24100054

  19. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    PubMed

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients.

  20. Cytokeratin19 induced by HER2/ERK binds and stabilizes HER2 on cell membranes

    PubMed Central

    Ju, J-h; Oh, S; Lee, K-m; Yang, W; Nam, K S; Moon, H-G; Noh, D-Y; Kim, C G; Park, G; Park, J B; Lee, T; Arteaga, C L; Shin, I

    2015-01-01

    Cytokeratin19 (KRT19) is widely used as a biomarker for the detection of disseminated tumors. Using an LC-MS/MS proteomics approach, we found that KRT19 was upregulated in HER2-overexpressing cells and tissues. KRT19 expression was induced by HER2-downstream ERK at the transcriptional level. Another HER2-downstream kinase, Akt, was found to phosphorylate KRT19 on Ser35 and induce membrane translocation of KRT19 and remodeling of KRT19 from filamentous to granulous form. KRT19 phosphorylated by Akt could bind HER2 on the plasma membrane and stabilized HER2 via inhibition of proteasome-mediated degradation of HER2. Silencing of KRT19 by shRNA resulted in increased ubiquitination and destabilization of HER2. Moreover, treatment of KRT19 antibody resulted in downregulation of HER2 and reduced cell viability. These data provide a new rationale for targeting HER2-positive breast cancers. PMID:25342465

  1. Infection with the Persistent Murine Norovirus Strain MNV-S99 Suppresses IFN-Beta Release and Activation of Stat1 In Vitro

    PubMed Central

    Niendorf, Sandra; Klemm, Uwe; Mas Marques, Andreas; Bock, C.-Thomas; Höhne, Marina

    2016-01-01

    Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-β production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-β release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response. PMID:27294868

  2. Interferon-gamma inhibits the neuronal differentiation of neural progenitor cells by inhibiting the expression of Neurogenin2 via the JAK/STAT1 pathway.

    PubMed

    Ahn, Jyhyun; Lee, Junsub; Kim, Sunyoung

    2015-10-01

    Interferon-gamma (IFN-γ) is one of the critical cytokines released by host immune cells upon infection. Despite the important role(s) of IFN-γ in host immune responses, there has been no in vivo study regarding the effects of IFN-γ on brain development, and the results from many in vitro studies are controversial. In this study, the effects of IFN-γ on embryonic neurogenesis were investigated. Treatment of E14.5 mouse neural progenitor cells (NPCs) with IFN-γ resulted in a decrease in the percentage of TuJ1-positive immature neurons but an increase in the percentage of Nestin-positive NPCs. Similar results were obtained in vivo. Treatment of NPCs with a JAK inhibitor or the knockdown of STAT1 expression abrogated the IFN-γ-mediated inhibition of neurogenesis. Interestingly, the expression of one of proneural genes, Neurogenin2 (Neurog2) was dramatically inhibited upon IFN-γ treatment, and cells overexpressing Neurog2 did not respond to IFN-γ. Taken together, our results demonstrate that IFN-γ inhibits neuronal differentiation of NPCs by negatively regulating the expression of Neurog2 via the JAK/STAT1 pathway. Our findings may provide an insight into the role of IFN-γ in the development of embryonic brain.

  3. The milk protein α-casein functions as a tumor suppressor via activation of STAT1 signaling, effectively preventing breast cancer tumor growth and metastasis

    PubMed Central

    Bonuccelli, Gloria; Castello-Cros, Remedios; Capozza, Franco; Martinez-Outschoorn, Ubaldo E.; Lin, Zhao; Tsirigos, Aristotelis; Xuanmao, Jiao; Whitaker-Menezes, Diana; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

    2012-01-01

    Here, we identified the milk protein α-casein as a novel suppressor of tumor growth and metastasis. Briefly, Met-1 mammary tumor cells expressing α-casein showed a ~5-fold reduction in tumor growth and a near 10-fold decrease in experimental metastasis. To identify the molecular mechanism(s), we performed genome-wide transcriptional profiling. Interestingly, our results show that α-casein upregulates gene transcripts associated with interferon/STAT1 signaling and downregulates genes associated with “stemness.” These findings were validated by immunoblot and FACS analysis, which showed the upregulation and hyperactivation of STAT1 and a decrease in the number of CD44(+) “cancer stem cells.” These gene signatures were also able to predict clinical outcome in human breast cancer patients. Thus, we conclude that a lactation-based therapeutic strategy using recombinant α-casein would provide a more natural and non-toxic approach to the development of novel anticancer therapies. PMID:23047602

  4. Protein stability induced by ligand binding correlates with changes in protein flexibility

    PubMed Central

    Celej, María Soledad; Montich, Guillermo G.; Fidelio, Gerardo D.

    2003-01-01

    The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted α-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA1,8-ANS, BSA2,6-ANS, BSAfree, BSAbis-ANS, and BSAunfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system. PMID:12824495

  5. The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

    PubMed

    Barbosa-Lorenzi, Valéria Cintra; Buranello, Patrícia Andressa de Almeida; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance; Pereira-da-Silva, Gabriela

    2011-12-16

    Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

  6. Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains.

    PubMed

    Zhao, K J; Chye, M L

    1999-08-01

    We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase. BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin: however there is only 36.9% identity between them. We propose that BjCHI1 should be classified under a new class, Chia7. The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain. Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyljasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA). This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA. Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B. juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge. Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E. coli JM109 expressing recombinant BjCHI1 showed chitinase activity. Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B. juncea. PMID:10527425

  7. From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

    PubMed Central

    Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

    2016-01-01

    Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

  8. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    PubMed

    Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

    2013-01-01

    ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.

  9. Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

    PubMed Central

    Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

    2013-01-01

    ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins

  10. Receptor-binding cancer antigen expressed on SiSo cells induces apoptosis via ectodomain shedding.

    PubMed

    Sonoda, Kenzo; Miyamoto, Shingo; Nakashima, Manabu; Wake, Norio

    2010-07-01

    Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a secreted antigen that induces apoptosis in putative receptor-expressing cells, including peripheral lymphocytes and natural killer (NK) cells. RCAS1 expression is associated with aggressive characteristics and poor overall survival for 15 different human malignancies. The putative RCAS1 receptor has not been isolated and the mechanism of RCAS1 apoptosis induction remains unclear. This study explores how RCAS1 is involved in apoptosis initiation. The cell lines SiSo and MCF-7, human uterine carcinoma and breast adenocarcinoma, respectively, both express RCAS1, but RCAS1 secretion is undetectable in MCF-7 cells. SiSo and MCF-7 cells were stimulated to induce RCAS1 ectodomain shedding followed by assessment of RCAS1 expression and secretion. Additionally, the RCAS1 putative receptor-expressing human chronic myelogenous leukemia cell line K562 was co-cultured with SiSo, MCF-7, or soluble RCAS1 to follow RCAS1 secretion in apoptosis initiation. RCAS1 secretion was strongly suppressed by inhibitors of metalloproteases, protein kinase C (PKC)-delta, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK), epidermal growth factor (EGF), and G-protein-coupled receptor (GPCR). K562 apoptosis could be induced only by co-culturing with SiSo or soluble RCAS1. RCAS1 is thus secreted by ectodomain shedding, which may represent a pivotal step in RCAS1-induced apoptosis initiation.

  11. Ca(2+)-induced folding and aggregation of skeletal muscle sarcoplasmic reticulum calsequestrin. The involvement of the trifluoperazine-binding site.

    PubMed

    He, Z; Dunker, A K; Wesson, C R; Trumble, W R

    1993-11-25

    Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.

  12. Cold-inducible RNA-binding protein is an important mediator of alcohol-induced brain inflammation.

    PubMed

    Rajayer, Salil R; Jacob, Asha; Yang, Weng-Lang; Zhou, Mian; Chaung, Wayne; Wang, Ping

    2013-01-01

    Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP(-/-)) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP(-/-) mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP(-/-) mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

  13. Cigarette smoke-induced reduction in binding of the salivary translocator protein is not mediated by free radicals.

    PubMed

    Nagler, R; Savulescu, D; Gavish, M

    2016-02-01

    Oral cancer is the most common malignancy of the head and neck and its main inducer is exposure to cigarette smoke (CS) in the presence of saliva. It is commonly accepted that CS contributes to the pathogenesis of oral cancer via reactive free radicals and volatile aldehydes. The 18 kDa translocator protein (TSPO) is an intracellular receptor involved in proliferation and apoptosis, and has been linked to various types of cancer. The presence of TSPO in human saliva has been linked to oral cancer, and its binding affinity to its ligand is reduced following exposure to CS. In the present study we wished to further investigate the mechanism behind the CS-induced reduction of TSPO binding by exploring the possible mediatory role of reactive oxygen species (ROS) and volatile aldehydes in this process. We first analyzed TSPO binding in control saliva and in saliva exposed to CS in the presence and absence of various antioxidants. These experiments found that TSPO binding ability was not reversed by any of the antioxidants added, suggesting that CS exerts its effect on TSPO via mechanisms that do not involve volatile aldehydes and free radicals tested. Next, we analyzed TSPO binding in saliva following addition of exogenous ROS in the form of H2O2. These experiments found that TSPO binding was enhanced due to the treatment, once again showing that the CS-induced TSPO binding reduction is not mediated by this common form of ROS. However, the previously reported CS-induced reduction in salivary TSPO binding together with the role of TSPO in cells and its link to cancer strongly suggest that TSPO has a critical role in the pathogenesis of CS-induced oral cancer. The importance of further elucidating the mechanisms behind it should be emphasized.

  14. An A-tract at the AtzR binding site assists DNA binding, inducer-dependent repositioning and transcriptional activation of the PatzDEF promoter.

    PubMed

    Porrúa, Odil; López-Sánchez, Aroa; Platero, Ana I; Santero, Eduardo; Shingler, Victoria; Govantes, Fernando

    2013-10-01

    The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF activation and high-affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR-induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A-tract sequence showed that the extent of AtzR-dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A-tract-associated narrow minor groove is essential for the AtzR-DNA complex to achieve a conformation competent for activation of the PatzDEF promoter. Conservation of this motif in several binding sites of LysR-type regulators suggests that this mechanism may be shared by other proteins in this family.

  15. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    PubMed

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  16. Structural Insight into the Role of Thrombospondin-1 Binding to Calreticulin in Calreticulin-Induced Focal Adhesion Disassembly†

    PubMed Central

    Yan, Qi; Murphy-Ullrich, Joanne E.; Song, Yuhua

    2010-01-01

    Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1–CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24–26 and 32–34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1–CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more “open” conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly. PMID:20337411

  17. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    PubMed

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering.

  18. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

    PubMed Central

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K.; Schlie, Katrin; Siebert, C. Alistair; Garten, Wolfgang; Bowden, Thomas A.; Strecker, Thomas; Huiskonen, Juha T.

    2016-01-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits. PMID:26849049

  19. Unique properties of cd-binding peptides induced in fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Hayashi, Y.; Nakagawa, C.W.; Murasugi, A.

    1986-03-01

    Metallothioneins, a class of low molecular weight cysteine-rich proteins that bind heavy metal ions, have been found in various eucaryotic organisms. When fission yeasts are grown in the presence of high concentration of CdCl/sub 2/, large amounts of Cd-binding peptides (Cd-BP1 and Cd-BP2) are synthesized. While Cd-BP2 shows similarities to mammalian Cd-thioneins in UV and CD spectra, Cd-BP1has a characteristic shoulder at 265 nm in the UV absorption spectrum and shows two marked Cotton bands at 257 nm (negative) and 275 nm (positive). These characteristics of Cd-BP1 are not found in the other Cd-thioneins. The UV and CD spectra differences between reconstituted and native Cd-BP1 suggest the requirement for some additional molecular architecture including another peptide-Cd/sup 2 +/ interaction. Induction of cadystin synthesis is almost exclusive for Cd, but an exception is a small amount of cadystin also induced by the higher concentration of CuCl/sub 2/ (2.5 mM). The UV spectrum of the natural Cu-cadystin complex was similar to that of Cd-BP1. On the basis of these findings the models for Cd-BP1 and Cd-BP2 are proposed.

  20. Spotted vesicles, striped micelles, and responsive Janus assemblies induced by ligand binding

    PubMed Central

    Christian, David A.; Tian, Aiwei; Ellenbroek, Wouter G.; Levental, Ilya; Rajagopal, Karthikan; Janmey, Paul A.; Liu, Andrea J.; Baumgart, Tobias; Discher, Dennis E.

    2009-01-01

    Selective binding of multivalent ligands within a mixture of polyvalent amphiphiles provides, in principle, a mechanism to drive domain formation in self-assemblies. Divalent cations are shown here to crossbridge polyanionic amphiphiles that thereby demix from neutral amphiphiles and form spots or rafts within vesicles as well as stripes within cylindrical micelles. Calcium and copper crossbridged domains of synthetic block copolymers or natural lipid (PIP2, phosphatidylinositol-4,5-bisphosphate) possess tunable sizes, shapes, and/or spacings that can last for years. Lateral segregation in these ‘responsive Janus assemblies’ couples weakly to curvature and proves restricted within phase diagrams to narrow regimes of pH and cation concentration that are centered near the characteristic binding constants for polyacid interactions. Remixing at high pH is surprising, but a theory for Strong Lateral Segregation (SLS) shows that counterion entropy dominates electrostatic crossbridges, thus illustrating the insights gained into ligand induced pattern formation within self-assemblies. PMID:19734886

  1. Tight-binding molecular dynamics simulations of radiation-induced C60 fragmentation

    NASA Astrophysics Data System (ADS)

    Beu, Titus A.; Horváth, Lóránd; Ghişoiu, Ioan

    2009-02-01

    The radiation-induced fragmentation of the C60 fullerene was investigated by tight-binding molecular dynamics simulations based on the parametrization of Papaconstantopoulos [Tight-Binding Approach to Computational Materials Science, edited by P.E.A. Turchi, A. Gonis, and L. Colombo, M.R.S. Symposia Proceedings No. 491 (Materials Research Society, Pittsburgh, 1998), p. 221] and employing novel models for nonadiabatic excitation and charge redistribution. The resulted fragment size and fragment charge distributions, averaged over large ensembles of trajectories corresponding to total ionization states up to +24e and excitation energies up to 1000 eV, have been used to analyze the fragmentation statistics in terms of several derived quantities. For moderate excitation energies, the fragment size profiles reproduce the experimentally observed U shape and bimodal dependence. Even though for high excitation energies and high total charges, predominantly multifragmentation occurs, a genuine power-law dependence sets in only beyond 1000 eV. A well-defined phase-transition region is found in the total charge-excitation energy plane, which appears to be delimited by a roughly parabolic critical line. The overall average critical excitation energy estimated from the simulations amounts to 55 eV and agrees with the experimental findings.

  2. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    PubMed

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K; Schlie, Katrin; Siebert, C Alistair; Garten, Wolfgang; Bowden, Thomas A; Strecker, Thomas; Huiskonen, Juha T

    2016-02-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  3. Hypertension induces tissue-specific gene suppression of a fatty acid binding protein in rat aorta.

    PubMed Central

    Sarzani, R; Claffey, K P; Chobanian, A V; Brecher, P

    1988-01-01

    The effect of hypertension on the expression of a fatty acid binding protein localized in the rat aorta was studied. The presence of rat heart fatty acid binding protein (hFABP) was documented in aortic tissue by using a cDNA probe and polyclonal antibodies. Hypertension was induced in groups of rats by implantation of deoxycorticosterone acetate in conjunction with 1% salt in the drinking water (deoxycorticosterone/salt). By the third week of this treatment a marked reduction (by a factor of 20) in the expression of hFABP mRNA in aorta was found, concomitant with a reduction in immunologically detectable protein, suggesting transcriptional regulation. This effect was tissue specific, since no change in the normal amounts of hFABP mRNA in heart, skeletal muscle, or kidney was found. This reduction in aortic hFABP mRNA was also found in mildly hypertensive uninephrectomized rats given salt but no deoxycorticosterone and in normotensive rats given deoxycorticosterone but no excess salt intake. A marked decrease in aortic hFABP mRNA also was observed in the Goldblatt two kidney-one clip hypertensive model, and administration of angiotensin II for 6 days by osmotic minipump also caused a reduction. These findings suggest that hFABP is under complex regulation in aortic tissue and is suppressed by arterial hypertension. Images PMID:3174661

  4. Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor

    SciTech Connect

    Raymond, Marc-Andre; Desormeaux, Anik; Labelle, Andree; Soulez, Mathilde; Soulez, Gilles; Langelier, Yves; Pshezhetsky, Alexey V.; Hebert, Marie-Josee . E-mail: marie-josee.hebert.chum@ssss.gouv.qc.ca

    2005-12-23

    Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1{alpha},25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.

  5. Libby amphibole-induced mesothelial cell autoantibodies bind to surface plasminogen and alter collagen matrix remodeling.

    PubMed

    Hanson, Robert; Evilia, Caryn; Gilmer, John; Woods, Linda; Black, Brad; Flores, Raja; Pfau, Jean C

    2016-08-01

    Lamellar pleural thickening (LPT) is a fibrotic disease induced by exposure to Libby amphibole (LA) asbestos that causes widespread scarring around the lung, resulting in deterioration of pulmonary function. Investigating the effects of autoantibodies to mesothelial cells (MCAA) present in the study populations has been a major part of the effort to understand the mechanism of pathogenesis. It has been shown in vitro that human mesothelial cells (Met5a) exposed to MCAA increase collagen deposition into the extracellular matrix (ECM). In this study, we sought to further elucidate how MCAA drive increased collagen deposition by identifying the protein targets bound by MCAA on the cellular surface using biotinylation to label and isolate surface proteins. Isolated surface protein fractions were identified as containing MCAA targets using ELISA The fractions that demonstrated binding by MCAA were then analyzed by tandem mass spectrometry (MS/MS) and MASCOT analysis. The most promising result from the MASCOT analysis, plasminogen (PLG), was tested for MCAA binding using purified human PLG in an ELISA We report that serum containing MCAA bound at an optical density (OD) 3 times greater than that of controls, and LA-exposed subjects had a high frequency of positive tests for anti-PLG autoantibodies. This work implicates the involvement of the plasminogen/plasmin system in the mechanism of excess collagen deposition in Met5a cells exposed to MCAA Elucidating this mechanism could contribute to the understanding of LPT. PMID:27519611

  6. Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

    PubMed

    Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

    2005-07-19

    The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

  7. Tight-binding molecular dynamics simulations of radiation-induced fragmentation of C60

    NASA Astrophysics Data System (ADS)

    Horváth, Lóránd; Beu, Titus A.

    2008-02-01

    The fragmentation of the C60 fullerene was investigated using tight-binding molecular dynamics simulations based on the parametrization of Papaconstantopoulos [MRS Symposia Proceedings No. 491 (Materials Research Society, Pittsburgh, 1998), p. 221]. Averaged fragment size distributions over random sets of initial configurations were obtained from simulations of radiation-induced fragmentation in the 50-500eV excitation energy range. The excitation caused by the radiation was simulated simply by ascribing suddenly random velocities to each atom of the fullerene cage. For low excitation energies, the size distributions are peaked for dimers (reflecting a preferential C2 emission) and a bimodal size dependence characterizes the distributions of the complementary small and large fragments. For high excitation energies, predominantly multifragmentation occurs, but a genuine power-law dependence of small fragments is not yet observable. A phase transition is found for rather low excitation energies (100-120eV) .

  8. Logic nanoparticle beacon triggered by the binding-induced effect of multiple inputs.

    PubMed

    Yang, Jing; Dong, Chen; Dong, Yafei; Liu, Shi; Pan, Linqiang; Zhang, Cheng

    2014-08-27

    Recently, the toehold-mediated DNA strand displacement reaction has been widely used in detecting molecular signals. However, traditional strand displacement, without cooperative signaling among DNA inputs, is insufficient for the design of more complicated nanodevices. In this work, a logic computing system is established using the cooperative "binding-induced" mechanism, based on the AuNP-based beacons, in which five kinds of multiple-input logic gates have been constructed. This system can recognize DNA and protein streptavidin simultaneously. Finally, the manipulations of the logic system are also demonstrated by controlling programmed conjugate DNA/AuNP clusters. This study provides the possibility of detecting multiple input signals and designing complex nanodevices that can be potentially applied to the detection of multiple molecular targets and the construction of large-scale DNA-based computation.

  9. Optimization of a Fragment-Based Screening Hit toward Potent DOT1L Inhibitors Interacting in an Induced Binding Pocket.

    PubMed

    Scheufler, Clemens; Möbitz, Henrik; Gaul, Christoph; Ragot, Christian; Be, Céline; Fernández, César; Beyer, Kim S; Tiedt, Ralph; Stauffer, Frédéric

    2016-08-11

    Mixed lineage leukemia (MLL) gene rearrangement induces leukemic transformation by ectopic recruitment of disruptor of telomeric silencing 1-like protein (DOT1L), a lysine histone methyltransferase, leading to local hypermethylation of H3K79 and misexpression of genes (including HoxA), which drive the leukemic phenotype. A weak fragment-based screening hit identified by SPR was cocrystallized with DOT1L and optimized using structure-based ligand optimization to yield compound 8 (IC50 = 14 nM). This series of inhibitors is structurally not related to cofactor SAM and is not interacting within the SAM binding pocket but induces a pocket adjacent to the SAM binding site.

  10. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    PubMed

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-01

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  11. A new generation of proto-oncogenes: cold-inducible RNA binding proteins.

    PubMed

    Lleonart, M E

    2010-01-01

    This review focuses on the roles of two major cold-inducible RNA binding proteins known in human cells: CIRP and RBM3. Both proteins were discovered when they were shown to be induced after exposure to a moderate cold-shock and other cellular stresses such as UV radiation and hypoxia. Initially, it was suggested that these proteins have a suppressive rather stimulatory effect on proliferation; however, proliferative and/or proto-oncogenic functions have recently been assigned to CIRP and RBM3. In a high throughput genetic screen, we recently identified CIRP as an immortalized gene in murine primary cells. On the other hand, the role of RBM3 in transformation has already been demonstrated. Interestingly, both CIRP and RBM3 have been found to be up-regulated in human tumors. This article highlights the roles of CIRP and RBM3 in tumorigenesis, and proposes a model by which CIRP might contribute to senescence bypass by counteracting the deleterious effects of oxidative damage.

  12. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival

    PubMed Central

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21WAF-1 protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation. PMID:27441652

  13. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival.

    PubMed

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21(WAF-1) protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation. PMID:27441652

  14. The role of interstitial binding in radiation induced segregation in W-Re alloys

    NASA Astrophysics Data System (ADS)

    Gharaee, Leili; Marian, Jaime; Erhart, Paul

    2016-07-01

    Due to their high strength and advantageous high-temperature properties, tungsten-based alloys are being considered as plasma-facing candidate materials in fusion devices. Under neutron irradiation, rhenium, which is produced by nuclear transmutation, has been found to precipitate in elongated precipitates forming thermodynamic intermetallic phases at concentrations well below the solubility limit. Recent measurements have shown that Re precipitation can lead to substantial hardening, which may have a detrimental effect on the fracture toughness of W alloys. This puzzle of sub-solubility precipitation points to the role played by irradiation induced defects, specifically mixed solute-W interstitials. Here, using first-principles calculations based on density functional theory, we study the energetics of mixed interstitial defects in W-Re, W-V, and W-Ti alloys, as well as the heat of mixing for each substitutional solute. We find that mixed interstitials in all systems are strongly attracted to each other with binding energies of -2.4 to -3.2 eV and form interstitial pairs that are aligned along parallel first-neighbor <111 > strings. Low barriers for defect translation and rotation enable defect agglomeration and alignment even at moderate temperatures. We propose that these elongated agglomerates of mixed-interstitials may act as precursors for the formation of needle-shaped intermetallic precipitates. This interstitial-based mechanism is not limited to radiation induced segregation and precipitation in W-Re alloys but is also applicable to other body-centered cubic alloys.

  15. DNA binding induces conformational transition within human DNA topoisomerase I in solution.

    PubMed

    Oleinikov, Vladimir; Sukhanova, Alyona; Mochalov, Konstantin; Ustinova, Olga; Kudelina, Irina; Bronstein, Igor; Nabiev, Igor

    2002-01-01

    We employed Raman and circular dichroism (CD) spectroscopy to probe the molecular structure of 68-kDa recombinant human DNA topoisomerase I (TopoI) in solution, in a complex with a 16-bp DNA fragment containing a camptothecin-enhanced TopoI cleavage site, and in a ternary complex with this oligonucleotide and topotecan. Raman spectroscopy reveals a TopoI secondary structure transition and significant changes in the hydrogen bonding of the tyrosine residues induced by the DNA binding. CD spectroscopy confirms the Raman data and identifies a DNA-induced (>7%) decrease of the TopoI alpha helix accompanied by at least a 6% increase of the beta structure. The Raman DNA molecular signatures demonstrated a bandshift that is expected for a net change in the environment of guanine C6 [double bond] O groups from pairing to solvent exposure. The formation of a ternary cleavage complex with TopoI, DNA, and topotecan as probed by CD spectroscopy reveals neither additional modifications of the TopoI secondary structure nor of the oligonucleotide structure, compared to the TopoI-oligonucleotide complex. PMID:12209444

  16. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival.

    PubMed

    Fujiwara, T; Zhou, J; Ye, S; Zhao, H

    2016-01-01

    The Musashi family of RNA-binding proteins, Musashi1 and Musashi2, regulate self-renewal and differentiation of neuronal and hematopoietic stem cells by modulating protein translation. It has been recently reported that Musashi2, not Musashi1, regulates hematopoietic stem cells. Although osteoclasts are derived from hematopoietic cells, the expression and functions of Musashi proteins in osteoclast lineage cells remain unknown. In this study, we have uncovered that Musashi2 is the predominant isoform of Musashi proteins in osteoclast precursors and its expression is upregulated by receptor activator of NF-κB ligand (RANKL) during osteoclast differentiation. Knocking down the expression of Musashi2 in osteoclast lineage cells by shRNAs attenuates nuclear factor of activated T cells 1 (NFATc1) expression and osteoclast formation in vitro. Mechanistically, loss of Musashi2 inhibits Notch signaling during osteoclast differentiation and induces apoptosis in pre-osteoclasts. In contrast, depletion of Musashi2 has no effects on cell cycle progression and p21(WAF-1) protein expression in macrophages. Furthermore, depletion of Notch2 and its downstream target Hes1 in osteoclast precursors by shRNAs abrogates osteoclastogenesis by inhibiting NFATc1. Finally, absence of Musashi2 in osteoclast precursors promotes apoptosis and inhibits RANKL-induced nuclear factor-κB (NF-κB) activation, which is essential for osteoclast survival, Thus, Musashi2 is required for cell survival and optimal osteoclastogenesis by affecting Notch signaling and NF-κB activation.

  17. Reduction of GABA/sub B/ receptor binding induced by climbing fiber degeneration in the rat cerebellum

    SciTech Connect

    Kato, K.; Fukuda, H.

    1985-07-22

    When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABA/sub B/ receptor binding determined with /sup 3/H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the B/sub max/ of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABA/sub B/ sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABA/sub A/ and benzodiazepine receptor binding labelled with /sup 3/H-muscimol and /sup 3/H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABA/sub B/ sites but neither GABA/sub A/ nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals. 28 references, 4 figures, 2 tables.

  18. Quantitative characterization of heparin binding to Tau protein: implication for inducer-mediated Tau filament formation.

    PubMed

    Zhu, Hai-Li; Fernández, Cristina; Fan, Jun-Bao; Shewmaker, Frank; Chen, Jie; Minton, Allen P; Liang, Yi

    2010-02-01

    Neurofibrillary tangles, principally composed of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer disease. Polyanionic cofactors such as heparin can induce Tau filament formation in vitro. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau(244-372) and heparin (average molecular mass = 7 kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays, and transmission electron microscopy. Our data clearly show that at physiological pH, heparin 7K, and human Tau(244-372) form a tight 1:1 complex with an equilibrium association constant exceeding 10(6) m(-1) under reducing conditions, triggering Tau fibrillization. In the absence of dithiothreitol, heparin shows a moderate binding affinity (10(5) m(-1)) to Tau(244-372), similarly triggering Tau fibrillization. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of 2:1 and then increases at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau and then decreases to some extent with ratios of >1:1. The retarding effect of heparin in excess is attributed to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of the 1:1 complex of Tau monomer and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases.

  19. Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

    PubMed

    Tan, Cheng; Li, Wenfei; Wang, Wei

    2013-12-19

    Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

  20. Synthesis and biological evaluation of benzimidazole acridine derivatives as potential DNA-binding and apoptosis-inducing agents.

    PubMed

    Gao, Chunmei; Li, Bin; Zhang, Bin; Sun, Qinsheng; Li, Lulu; Li, Xi; Chen, Changjun; Tan, Chunyan; Liu, Hongxia; Jiang, Yuyang

    2015-04-15

    The discovery of new effective DNA-targeted antitumor agent is needed because of their clinical significance. As acridines can intercalate into DNA and benzimidazoles have the ability to bind in the DNA minor groove, a series of novel benzimidazole acridine derivatives were designed and synthesized to be new DNA-targeted compounds. MTT assay indicated that most of the synthesized compounds displayed good antiproliferative activity, among which compound 8l demonstrated the highest activity against both K562 and HepG-2 cells. Further experiments showed that 8l displayed good DNA-binding capability and inhibited topoisomerase I activity. Moreover, compound 8l could induce apoptosis in K562 cell lines through mitochondrial pathway. These data suggested that compound 8l might be potential as new DNA-binding and apoptosis-inducing antitumor agents.

  1. A fibrin antibody binding to fibronectin induces potent inhibition of angiogenesis.

    PubMed

    El-Ayoubi, Fida; Amiral, Jean; Pascaud, Juliette; Charrin, Stéphanie; Tassel, Bénédicte; Uzan, Georges; Gurewich, Victor

    2015-01-01

    Antiserum from rabbits immunised with pure human fibrinogen was affinity purified on immobilised fibrin fragment E (FFE). This FFE antibody (Ab) induced significant growth inhibition of a human cancer xenograft in mice and suppression of tumour angiogenesis, leaving no formed vessels and only CD31-staining endothelial fragments in place. Tubule formation of HUVEC on MatrigelTM was also significantly inhibited by FFE Ab. Since MatrigelTM is fibrin-free, this effect implicated a different FFE Ab binding site than FFE. Flow cytometry of HUVEC showed that FFE Ab bound to HUVEC, but with a broad range of 55-98 %. Immunofluorescent staining of HUVEC explained this range, since FFE Ab was seen not to bind to human umbilical vein endothelial cells (HUVEC) directly but instead to a matrix protein variably adherent to HUVEC. This protein was identified as fibronectin (FN) by appearance, staining with FN Ab, and by a FN knockdown study. Neither HUVEC nor matrix reacted with fibrin D-dimer (DD) Ab. Immunofluorescent stains of HUVEC matrix with FFE and FN Ab's showed that these Ab's bound to the same epitopes on FN, as also seen on Western blots of purified FN. These findings indicate the presence of an antigenic determinant in fibrinogen/FFE that is homologous with an epitope(s) in FN recognised by FFE Ab, and critical for angiogenesis in this xenograft. The FN epitope(s) remains to be identified, but the present findings can be used for the selection of the appropriate clones from mice immunised with fibrinogen which can facilitate this identification, and which may also be of clinical use. PMID:25252851

  2. Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1.

    PubMed

    Oi, N; Yuan, J; Malakhova, M; Luo, K; Li, Y; Ryu, J; Zhang, L; Bode, A M; Xu, Z; Li, Y; Lou, Z; Dong, Z

    2015-05-14

    Resveratrol (trans-3,5,4'-truhydroxystilbene) possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to the suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1.

  3. Role of vitamin D-binding protein in isocyanate-induced occupational asthma.

    PubMed

    Kim, Sung-Ho; Choi, Gil-Soon; Nam, Young-Hee; Kim, Joo-Hee; Hur, Gyu-Young; Kim, Seung-Hyun; Park, Sang Myun; Park, Hae-Sim

    2012-05-31

    The development of a serological marker for early diagnosis of isocyanate-induced occupational asthma (isocyanate-OA) may improve clinical outcome. Our previous proteomic study found that expression of vitamin D-binding protein (VDBP) was upregulated in the patients with isocyanate-OA. In the present study, we evaluated the clinical relevance of VDBP as a serological marker in screening for isocyanate-OA among exposed workers and its role in the pathogenesis of isocyanate-OA. Three study groups including 61 patients with isocyanate-OA (group I), 180 asymptomatic exposed controls (AECs, group II), 58 unexposed healthy controls (NCs, group III) were enrolled in this study. The baseline serum VDBP level was significantly higher in group I compared with groups II and III. The sensitivity and specificity for predicting the phenotype of isocyanate-OA with VDBP were 69% and 81%, respectively. The group I subjects with high VDBP (≥311 μg/ml) had significantly lower PC(20) methacholine levels than did subjects with low VDBP. The in vitro studies showed that TDI suppressed the uptake of VDBP into RLE-6TN cells, which was mediated by the downregulation of megalin, an endocytic receptor of the 25-hydroxycholecalciferol-VDBP complex. Furthermore, toluene diisocyanate (TDI) increased VEGF production and secretion from this epithelial cells by suppression of 1,25-dihydroxycholecalciferol [1,25(OH)(2)D(3)] production. The findings of this study suggest that the serum VDBP level may be used as a serological marker for the detection of isocyanate-OA among workers exposed to isocyanate. The TDI-induced VEGF production/ secretion was reversed by 1,25(OH)(2)D(3) treatment, which may provide a potential therapeutic strategy for patients with isocyanate-OA.

  4. Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins

    PubMed Central

    Al-Zeer, Munir A.; Al-Younes, Hesham M.; Lauster, Daniel; Abu Lubad, Mohammad; Meyer, Thomas F.

    2013-01-01

    Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance. PMID:23086406

  5. Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.

    PubMed Central

    Parker, D; Ferreri, K; Nakajima, T; LaMorte, V J; Evans, R; Koerber, S C; Hoeger, C; Montminy, M R

    1996-01-01

    We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids. PMID:8552098

  6. Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding.

    PubMed

    Miki, M; dos Remedios, C G; Barden, J A

    1987-10-15

    intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.

  7. Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp

    PubMed Central

    Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

    2016-01-01

    In mammals, body temperature fluctuates diurnally around a mean value of 36°C–37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells’ circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide “approach to steady-state” kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

  8. Troponin I binds polycystin-L and inhibits its calcium-induced channel activation.

    PubMed

    Li, Qiang; Liu, Yan; Shen, Patrick Y; Dai, Xiao-Qing; Wang, Shaohua; Smillie, Lawrence B; Sandford, Richard; Chen, Xing-Zhen

    2003-06-24

    Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I. PMID:12809519

  9. Guanylate Binding Protein (GBP) 5 Is an Interferon-Inducible Inhibitor of HIV-1 Infectivity.

    PubMed

    Krapp, Christian; Hotter, Dominik; Gawanbacht, Ali; McLaren, Paul J; Kluge, Silvia F; Stürzel, Christina M; Mack, Katharina; Reith, Elisabeth; Engelhart, Susanne; Ciuffi, Angela; Hornung, Veit; Sauter, Daniel; Telenti, Amalio; Kirchhoff, Frank

    2016-04-13

    Guanylate binding proteins (GBPs) are an interferon (IFN)-inducible subfamily of guanosine triphosphatases (GTPases) with well-established activity against intracellular bacteria and parasites. Here we show that GBP5 potently restricts HIV-1 and other retroviruses. GBP5 is expressed in the primary target cells of HIV-1, where it impairs viral infectivity by interfering with the processing and virion incorporation of the viral envelope glycoprotein (Env). GBP5 levels in macrophages determine and inversely correlate with infectious HIV-1 yield over several orders of magnitude, which may explain the high donor variability in macrophage susceptibility to HIV. Antiviral activity requires Golgi localization of GBP5, but not its GTPase activity. Start codon mutations in the accessory vpu gene from macrophage-tropic HIV-1 strains conferred partial resistance to GBP5 inhibition by increasing Env expression. Our results identify GBP5 as an antiviral effector of the IFN response and may explain the increased frequency of defective vpu genes in primary HIV-1 strains. PMID:26996307

  10. Shear induced carboplatin binding within the cavity of a phospholipid mimic for increased anticancer efficacy.

    PubMed

    Mo, Jingxin; Eggers, Paul K; Chen, Xianjue; Ahamed, Muhammad Rizwan Hussain; Becker, Thomas; Yong Lim, Lee; Raston, Colin L

    2015-05-22

    Vesicles 107 ± 19 nm in diameter, based on the self-assembly of tetra-para-phosphonomethyl calix[4]- arene bearing n-hexyl moieties attached to the phenolic oxygen centres, are effective in binding carboplatin within the cavity of the macrocycle under shear induced within a dynamic thin film in a continuous flow vortex fluidic device. Post shearing the vesicles maintain similar diameters and retain carboplatin within the cavity of the calixarene in a hierarchical structure, with their size and morphology investigated using DLS, TEM, SEM and AFM. Location of the carboplatin was confirmed using NMR, FTIR, ESI-MS and EFTEM, with molecular modelling favouring the polar groups of carboplatin hydrogen bonded to phosphonic acid moieties and the four member cyclobutane ring directed into the cavity of the calixarene. The loading efficiency and release profile of carboplatin was investigated using LC-TOF/MS, with the high loading of the drug achieved under shear and preferential released at pH 5.5, offering scope for anti-cancer drug delivery. The hierarchical structured vesicles increase the efficacy of carboplatin by 4.5 fold on ovarian cancer cells, lowered the IC50 concentration by 10 fold, and markedly increased the percent of cells in the S-phase (DNA replication) of the cell cycle.

  11. Temperature regulates splicing efficiency of the cold-inducible RNA-binding protein gene Cirbp.

    PubMed

    Gotic, Ivana; Omidi, Saeed; Fleury-Olela, Fabienne; Molina, Nacho; Naef, Felix; Schibler, Ueli

    2016-09-01

    In mammals, body temperature fluctuates diurnally around a mean value of 36°C-37°C. Despite the small differences between minimal and maximal values, body temperature rhythms can drive robust cycles in gene expression in cultured cells and, likely, animals. Here we studied the mechanisms responsible for the temperature-dependent expression of cold-inducible RNA-binding protein (CIRBP). In NIH3T3 fibroblasts exposed to simulated mouse body temperature cycles, Cirbp mRNA oscillates about threefold in abundance, as it does in mouse livers. This daily mRNA accumulation cycle is directly controlled by temperature oscillations and does not depend on the cells' circadian clocks. Here we show that the temperature-dependent accumulation of Cirbp mRNA is controlled primarily by the regulation of splicing efficiency, defined as the fraction of Cirbp pre-mRNA processed into mature mRNA. As revealed by genome-wide "approach to steady-state" kinetics, this post-transcriptional mechanism is widespread in the temperature-dependent control of gene expression. PMID:27633015

  12. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

  13. Type II secretory phospholipase A2 binds to ischemic flip-flopped cardiomyocytes and subsequently induces cell death.

    PubMed

    Nijmeijer, R; Willemsen, M; Meijer, C J L M; Visser, C A; Verheijen, R H; Gottlieb, R A; Hack, C E; Niessen, H W M

    2003-11-01

    Type II secretory phospholipase A2 (sPLA2) is a cardiovascular risk factor. We recently found depositions of sPLA2 in the necrotic center of infarcted human myocardium and normally appearing cardiomyocytes adjacent to the border zone. The consequences of binding of sPLA2 to ischemic cardiomyocytes are not known. To explore a potential effect of sPLA2 on ischemic cardiomyocytes at a cellular level we used an in vitro model. The cardiomyocyte cell line H9c2 or adult cardiomyocytes were isolated from rabbits that were incubated with sPLA2 in the presence of metabolic inhibitors to mimic ischemia-reperfusion conditions. Cell viability was established with the use of annexin V and propidium iodide or 7-aminoactinomycin D. Metabolic inhibition induced an increase of the number of flip-flopped cells, including a population that did not stain with propidium iodide and that was caspase-3 negative. sPLA2 bound to the flip-flopped cells, including those negative for caspase-3. sPLA2 binding induced cell death in these latter cells. In addition, sPLA2 potentiated the binding of C-reactive protein (CRP) to these cells. We conclude that by binding to flip-flopped cardiomyocytes, including those that are caspase-3 negative and presumably reversibly injured, sPLA2 may induce cell death and tag these cells with CRP.

  14. Pain hypersensitivity induced by paradoxical sleep deprivation is not due to altered binding to brain mu-opioid receptors.

    PubMed

    Nascimento, Danielle C; Andersen, Monica L; Hipólide, Débora Cristina; Nobrega, José N; Tufik, Sergio

    2007-03-28

    Previous studies have established a relationship between sleep disruption and pain, and it has been suggested that hyperalgesia induced by paradoxical sleep deprivation (PSD) could be due to a reduction of opioidergic neurotransmission in the brain. In the present study rats deprived of sleep for 96 h as well as rats allowed to recover for 24h after PSD and normal controls received vehicle or morphine (2.5, 5 and 10 mg/kg, i.p.) and were tested on a hot plate 1h later. Quantitative receptor autoradiography was used to map alterations in binding to brain mu-opioid receptors in separate groups. Results demonstrated that PSD induced a significant reduction in thermal pain threshold, as measured by paw withdrawal latencies. This effect did not return to baseline control values after 24h of sleep recovery. The usual analgesic effect of morphine was observed in the control group but not in PSD or rebound groups except at the highest dose (10 mg/kg). Binding of [3H]DAMGO to mu sites did not differ significantly among the three groups in any of the 33 brain regions examined. These results do not exclude the participation of the opioid system in PSD-induced pain hypersensitivity since sleep-deprived rats were clearly resistant to morphine. However, the fact no changes were seen in [3H]DAMGO binding indicates that mechanisms other than altered mu-opioid binding must be sought to explain the phenomenon.

  15. von Willebrand factor binds to platelets and induces aggregation in platelet-type but not type IIB von Willebrand disease.

    PubMed Central

    Miller, J L; Kupinski, J M; Castella, A; Ruggeri, Z M

    1983-01-01

    Platelet-type von Willebrand disease (vWD) and pseudo-vWD are two recently described intrinsic platelet defects characterized by enhanced ristocetin-induced agglutination in platelet-rich plasma. A similar finding is also typical of type IIB vWD, where it has been related to a von Willebrand factor (vWF) rather than a platelet abnormality. Platelet aggregation induced by unmodified human vWF in the absence of other stimuli has been reported in pseudo-vWD. In this study we demonstrate that vWF induces aggregation in platelet-type but not type IIB vWD. Aggregation is observed when normal plasma cryoprecipitate or purified vWF are added to platelet-rich plasma. Cryoprecipitate also aggregates washed platelets, although at higher concentrations than required for platelet-rich plasma. Purified vWF, however, induces significant aggregation of washed platelets only when plasma is added. EDTA inhibits vWF-induced aggregation. Its effect can be overcome by calcium but much less effectively by magnesium ions. Unstimulated platelets in platelet-rich plasma from patients with platelet-type but not type IIB vWD bind 125I-vWF in a specific and saturable manner. All different sized multimers of vWF become associated with platelets. Both aggregation and binding exhibit a similar vWF concentration dependence, suggesting that a correlation exists between these two events. Removal of ADP by appropriate consuming systems is without effect upon such binding or upon vWF-induced aggregation. Thrombin-induced 125I-vWF binding to washed platelets is normal in platelet-type as well as type IIB vWD. These results demonstrate that a specific binding site for unmodified human vWF is exposed on unstimulated platelets in platelet-type vWD. The relatively high vWF concentrations required for aggregation and binding may explain the lack of significant in vivo aggregation and thrombocytopenia in these patients. Moreover, these studies provide additional evidence that platelet-type and type IIB v

  16. Multi-scaled explorations of binding-induced folding of intrinsically disordered protein inhibitor IA3 to its target enzyme.

    PubMed

    Wang, Jin; Wang, Yong; Chu, Xiakun; Hagen, Stephen J; Han, Wei; Wang, Erkang

    2011-04-01

    Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change - protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA) by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.

  17. Low protein-high carbohydrate diet induces alterations in the serum thyronine-binding proteins in the rat.

    PubMed

    Young, R A; Braverman, L E; Rajatanavin, R

    1982-05-01

    The serum T3 concentration was increased in 8-week-old lean Zucker rats fed a low protein-high carbohydrate diet for 2 weeks. This increase was secondary to the generation of a binding protein migrating in the postalbumin zone in polyacrylamide gel electrophoresis employing 125I-labeled T3 and is termed rat thyronine-binding globulin. The presence of this T3-binding protein in serum resulted in a marked decrease in the percent free T3 assessed by equilibrium dialysis and a normal free T3 concentration. An increase in the binding of T4 in the postalbumin zone was also observed, but no changes in the dialyzable fraction of T4 or the total and free T4 concentrations occurred. In contrast to these findings in lean Zucker rats fed the low protein-high carbohydrate diet, no change in the pattern of 125I-labeled T3 and T4 binding, the dialyzable fraction of T3 or T4, or total and free T3 or T4 concentrations were observed in the obese Zucker rats fed this diet. The present findings suggest that diet-induced alterations in thyroid hormone-binding proteins must be considered in the interpretation of data which involve alterations in total thyroid hormone concentrations in serum and their role in affecting tissue metabolism.

  18. Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction.

    PubMed Central

    Yan, H; Krishnan, K; Lim, J T; Contillo, L G; Krolewski, J J

    1996-01-01

    Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction. PMID:8628273

  19. Aptamer-based electrochemical sensors that are not based on the target binding-induced conformational change of aptamers.

    PubMed

    Lu, Ying; Zhu, Ningning; Yu, Ping; Mao, Lanqun

    2008-09-01

    This study describes a new kind of aptamer-based electrochemical sensor that is not based on the target binding-induced conformational change of the aptamers by using a 15-mer thrombin-binding aptamer (5'-GGTTGGTGTGGTTGG-3') as the model oligonucleotide. The sensors are developed by first self-assembling the aptamer (i.e. a thrombin-binding aptamer) onto an Au electrode and then hybridizing the assembled aptamer with a ferrocene (Fc)-labeled short aptamer-complementary DNA oligonucleotide to form an electroactive double-stranded DNA (ds-DNA) oligonucleotide onto the Au electrode. The binding of the target (i.e. thrombin) towards the aptamer essentially destroys the Watson-Crick helix structure of the ds-DNA oligonucleotide assembled onto the electrode and leads to the dissociation of the Fc-labeled short complementary DNA oligonucleotide from the electrode surface to the solution, resulting in a decrease in the current signal obtained at the electrode, which can be used for the determination of the target. With the thrombin-binding aptamer as the model oligonucleotide, the current decrease obtained with the aptamer-based electrochemical sensors is linear with the concentration of thrombin within the concentration range from 0 to 10 nM (DeltaI/nA = 6.7C(thrombin)/nM + 2.8, gamma = 0.975). Unlike most kinds of existing aptamer-based electrochemical sensor, the electrochemical aptasensors demonstrated here are not based on the conformational change of the aptamers induced by the specific target binding. Moreover, the aptasensors are essentially label-free and are very responsive toward the targets. This study may pave a facile and general way to the development of aptamer-based electrochemical sensors.

  20. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    PubMed

    Norris, M L; Millhorn, D E

    1995-10-01

    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  1. Theoretical studies on the binding of rhenium(I) complexes to inducible nitric oxide synthase.

    PubMed

    Oliveira, Bruno L; Moreira, Irina S; Fernandes, Pedro A; Ramos, Maria J; Santos, Isabel; Correia, João D G

    2013-09-01

    Considering our interest in the design of innovative radiometal-based complexes for in vivo imaging of nitric oxide synthase (NOS), we have recently introduced a set of M(CO)3-complexes (M=(99m)Tc, Re) containing a pendant N(ω)-NO2-L-arginine moiety, a known inhibitor of the enzyme. Enzymatic assays with purified inducible NOS have shown that the non-radioactive surrogates with 3-(Re1; Ki=84 μM) or 6-carbon linkers (Re2; Ki=6 μM) are stronger inhibitors than the respective metal-free conjugates L1 (Ki=178 μM) and L2 (Ki=36 μM), with Re2 displaying the highest inhibitory potency. Aiming to rationalize the experimental results we have performed a molecular docking study combined with molecular dynamics (MD) simulations and free energy perturbation (FEP) calculations. The higher inhibitory potency of Re2 arises from the stronger electrostatic interactions observed between the "Re(CO)3" core and the residues Arg260 and Arg382. This interaction is only possible due to the higher flexibility of its C6-carbon spacer, which links the N(ω)-NO2-L-arginine moiety and the "Re(CO)3" organometallic core. Furthermore, FEP calculations were carried out and the resultant relative binding energies (ΔΔGbind(calc)=0.690±0.028 kcal/mol,Re1/L1 and 1.825±0.318 kcal/mol, Re2/L2) are in accordance with the experimental results (ΔΔGbind(exp)=0.461±0.009 kcal/mol,Re1/L1 and 1.129±0.210 kcal/mol, Re2/L2); there is an energetic penalty for the transformation of the Re complexes into the ligands and this penalization is higher for the pair Re2/L2.

  2. DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis.

    PubMed

    Iuchi, Katsuya; Yagura, Tatsuo

    2016-03-15

    Ku protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku protein during apoptosis remains unclear. Here, we show characterization of Ku protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the non-treated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion, Ku is intact and retains DNA binding activity in early apoptotic cells.

  3. A synchrotron X-ray scattering characterization of purified tubulin and of its expansion induced by mild detergent binding

    SciTech Connect

    Andreu, J.M.; de Ancos, J.G.; Starling, D.; Hodgkinson, J.L.; Bordas, J. )

    1989-05-02

    This report presents a synchrotron radiation X-ray scattering characterization of calf brain tubulin purified by the modified Weisenberg procedure. The results show that under nonassembly conditions these preparations consist of a uniform population of molecules with a radius of gyration of 3.1 {plus minus} 0.1 nm, which can be interpreted as arising from the native {alpha}-{beta} heterodimer. The uniformity in the population persists even at unusually high concentrations of protein. Binding of colchicine or substitution of GTP by GDP does not induce, within the statistical accuracy and resolution range of our measurements, any significant structural modification in soluble tubulin. In assembly buffer, these preparations readily assemble into microtubules upon increasing the temperature from 4 to 37{degree}C. Binding of nondenaturing amphiphiles to soluble tubulin provides a simplified model for tubulin-membrane interactions. The X-ray scattering data show that the radius of gyration of tubulin progressively increases upon binding of the mild detergent sodium deoxycholate, reaching a maximum value of 4.3 {plus minus} 0.1 nm at detergent saturation. The relative increase in the radius of gyration coincides within experimental error with the previously determined relative increase in the frictional coefficient. Analysis of these observations suggests that the effect of detergent binding is to induce an isotropic swelling of the protein structure.

  4. Conformational Selection and Induced Fit Mechanisms in the Binding of an Anticancer Drug to the c-Src Kinase

    PubMed Central

    Morando, Maria Agnese; Saladino, Giorgio; D’Amelio, Nicola; Pucheta-Martinez, Encarna; Lovera, Silvia; Lelli, Moreno; López-Méndez, Blanca; Marenchino, Marco; Campos-Olivas, Ramón; Gervasio, Francesco Luigi

    2016-01-01

    Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called “DFG-flip” of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an “in to out” movement resulting in a particular inactive conformation to which “type II” kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed. PMID:27087366

  5. Conformational Selection and Induced Fit Mechanisms in the Binding of an Anticancer Drug to the c-Src Kinase

    NASA Astrophysics Data System (ADS)

    Morando, Maria Agnese; Saladino, Giorgio; D’Amelio, Nicola; Pucheta-Martinez, Encarna; Lovera, Silvia; Lelli, Moreno; López-Méndez, Blanca; Marenchino, Marco; Campos-Olivas, Ramón; Gervasio, Francesco Luigi

    2016-04-01

    Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called “DFG-flip” of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an “in to out” movement resulting in a particular inactive conformation to which “type II” kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.

  6. Characterization of the species-dependent ketoprofen/albumin binding modes by induced CD spectroscopy and TD-DFT calculations.

    PubMed

    Tedesco, Daniele; Pistolozzi, Marco; Zanasi, Riccardo; Bertucci, Carlo

    2015-08-10

    The stereospecificity of high-affinity biorecognition phenomena at the basis of the activity of drugs is an important topic of active research in medicinal chemistry. The binding of drugs to their targets or to carrier proteins may lead to the onset of an induced circular dichroism (ICD) signal, which can be detected experimentally. Quantum mechanical (QM) calculations based on density functional theory (DFT) and its time-dependent formulation (TD-DFT) can be used to determine the theoretical chiroptical response of all the possible conformations of drugs bound to their hosts; by comparison with the experimental ICD spectra of drug-host complexes, this approach can lead to the identification of possible binding modes in the absence of X-ray crystallography or NMR data. The present article reports the application of experimental electronic circular dichroism (ECD) spectroscopy, DFT conformational analysis and TD-DFT calculations to the investigation of the binding modes of (S)-ketoprofen to serum albumins. The peculiar species-dependent ICD spectra observed for the binding of (S)-ketoprofen to different serum albumins can be explained by the selection of different mutual arrangements of the phenyl moieties inside the binding pocket. Such structural elucidations contribute to a better understanding of the changes in the pharmacokinetic and pharmacodynamic profiles of drugs among different species.

  7. Anion-induced increases in the affinity of colcemid binding to tubulin.

    PubMed

    Ray, K; Bhattacharyya, B; Biswas, B B

    1984-08-01

    Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.

  8. Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4.

    PubMed

    Mizuguchi, Chiharu; Hata, Mami; Dhanasekaran, Padmaja; Nickel, Margaret; Phillips, Michael C; Lund-Katz, Sissel; Saito, Hiroyuki

    2012-07-17

    Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).

  9. Secreted glypican binds to the amyloid precursor protein of Alzheimer's disease (APP) and inhibits APP-induced neurite outgrowth.

    PubMed

    Williamson, T G; Mok, S S; Henry, A; Cappai, R; Lander, A D; Nurcombe, V; Beyreuther, K; Masters, C L; Small, D H

    1996-12-01

    The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

  10. Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-{alpha}-induced NF-{kappa}B activity by binding to TNFR1

    SciTech Connect

    Hwang, Jae Ryoung; Huh, Jae Ho; Lee, Yoonna; Lee, Sang Il; Rho, Seung Bae; Lee, Je-Ho

    2011-02-25

    Research highlights: {yields} Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. {yields} The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-{alpha} and was blocked TNF-{alpha}-activated NF-{kappa}B activity. {yields} IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-{alpha}. {yields} Competition between the L-domain of IGFBP-5 and TNF-{alpha} blocked TNF-{alpha}-induced NF-{kappa}B activity. {yields} This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor. -- Abstract: IGFBP-5 is known to be involved in various cell phenomena such as proliferation, differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-{alpha}, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-{kappa}B activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-{alpha} in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-{alpha} resulted in inhibition of TNF-{alpha}-induced NF-{kappa}{Beta} activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF

  11. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha.

    PubMed

    Yuan, Yong; Hilliard, George; Ferguson, Tsuneo; Millhorn, David E

    2003-05-01

    The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The alpha-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-alpha (including HIF-1alpha and HIF-2alpha). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-alpha is required for the interaction between HIF and VHL. It has previously been established that cobalt mimics hypoxia and causes accumulation of HIF-1alpha and HIF-2alpha. However, little is known about the mechanism by which this occurs. In an earlier study, we demonstrated that cobalt binds directly to the ODD domain of HIF-2alpha. Here we provide the first evidence that cobalt inhibits pVHL binding to HIF-alpha even when HIF-alpha is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2alpha that are required for pVHL binding prevented the binding of cobalt and stabilized HIF-2alpha during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-alpha and thereby preventing the degradation of HIF-alpha. PMID:12606543

  12. Binding to the lipid monolayer induces conformational transition in Aβ monomer.

    PubMed

    Kim, Seongwon; Klimov, Dmitri K

    2013-02-01

    Using implicit solvent atomistic model and replica exchange molecular dynamics, we study binding of Aβ monomer to zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid monolayer. Our results suggest that Aβ binding to the monolayer is governed primarily by positively charged and aromatic amino acids. Lysine residues tend to interact with surface choline and phosphorous lipid groups, whereas aromatic amino acids penetrate deeper into the monolayer, reaching its hydrophobic core. We show that binding to the DMPC monolayer causes a dramatic conformational transition in Aβ monomer, resulting in chain extension, loss of intrapeptide interactions, and formation of β-structure. This conformational transition is far more significant than that occurring during the initial stages of aggregation in water. We also found that Aβ binding perturbs surface ordering of lipids interacting with Aβ. PMID:23053007

  13. Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)*

    PubMed Central

    Benned-Jensen, Tau; Norn, Christoffer; Laurent, Stephane; Madsen, Christian M.; Larsen, Hjalte M.; Arfelt, Kristine N.; Wolf, Romain M.; Frimurer, Thomas; Sailer, Andreas W.; Rosenkilde, Mette M.

    2012-01-01

    Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors. PMID:22875855

  14. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    SciTech Connect

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.

  15. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  16. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  17. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  18. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

    PubMed

    Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

    1999-03-01

    The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors.

  19. FBI-1, a factor that binds to the HIV-1 inducer of short transcripts (IST), is a POZ domain protein.

    PubMed Central

    Morrison, D J; Pendergrast, P S; Stavropoulos, P; Colmenares, S U; Kobayashi, R; Hernandez, N

    1999-01-01

    The HIV-1 promoter directs the synthesis of two classes of transcripts, short, non-polyadenylated transcripts and full-length, polyadenylated transcripts. The synthesis of short transcripts is activated by a bipartite DNA element, the inducer of short transcripts or IST, located downstream of the HIV-1 transcriptional start site, while the synthesis of full-length transcripts is activated by the viral activator Tat. Tat binds to the RNA element TAR, which is encoded largely between the two IST half-elements. Upon activation by Tat, the synthesis of short RNAs is repressed. We have previously purified a factor called FBI-1 (for factor that binds to IST) whose binding to wild-type and mutated ISTs correlated well with the abilities of these ISTs to direct the synthesis of short transcripts. Here, we report the cloning of cDNAs encoding FBI-1. FBI-1 contains a POZ domain at its N-terminus and four Krüppel-type zinc fingers at its C-terminus. The C-terminus is sufficient for specific binding, and FBI-1 can form homomers through its POZ domain and, in vivo, through its zinc finger domain as well. In addition, FBI-1 associates with Tat, suggesting that repression of the short transcripts by Tat may be mediated through interactions between the two factors. PMID:9973611

  20. Electrochemical aptameric recognition system for a sensitive protein assay based on specific target binding-induced rolling circle amplification.

    PubMed

    Wu, Zai-Sheng; Zhou, Hui; Zhang, Songbai; Shen, Guoli; Yu, Ruqin

    2010-03-15

    A reusable aptameric recognition system was described for the electrochemical detection of the protein PDGF-BB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe, and primer probe were utilized to introduce a RCA process into the aptamer-target binding event while a new aptamer was elegantly designed via lengthening the original aptamer by the complement to the CDNA. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: a unique electrochemical technique, an attractive RCA process, reversible DNA hybridization, and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits excellent performance (e.g., a detection limit of 6.3 x 10(-11) M, a linear dynamic range of 2 orders of magnitude, high specificity, and satisfactory repeatability) but also overcomes the limitations associated with conventional aptameric biosensors (e.g., dependence of signaling target binding on specific aptamer sequence or requirement of sandwich assays for two or more binding sites per target molecule). A recovery test demonstrated the feasibility of the developed target protein assay. Given the attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research.

  1. Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage▿

    PubMed Central

    Alekseev, Sergey; Luijsterburg, Martijn S.; Pines, Alex; Geverts, Bart; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina; Lans, Hannes; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Hoeijmakers, Jan H. J.; Vermeulen, Wim

    2008-01-01

    Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions. PMID:18936169

  2. Increased /sup 3/H-spiperone binding sites in mesolimbic area related to methamphetamine-induced behavioral hypersensitivity

    SciTech Connect

    Akiyama, K.; Sato, M.; Otsuki, S.

    1982-02-01

    The specific /sup 3/H-spiperone binding to membrane homogenates of the striatum, mesolimbic area, and frontal cortex was examined in two groups of rats pretreated once daily with saline or 4 mg/kg of methamphetamine (MAP) for 14 days. At 7 days following cessation of chronic pretreatment, all rats received an injection of 4 mg/kg of MAP and were decapitated 1 hr after the injection. In the chronic saline-pretreatment group, the single administration of MAP induced significant changes in the number (Bmax) of specific /sup 3/H-spiperone binding sites (a decrease in the striatum and an increase in the mesolimbic area and frontal cortex), but no significant changes in the affinity (KD) in any brain area. The chronic MAP pretreatment markedly augmented the changes in Bmax in the striatum and mesolimbic area. The increase in specific /sup 3/H-spiperone binding sites in the mesolimbic area is discussed in relation to MAP-induced behavioral hypersensitivity.

  3. Zinc Induces Dimerization of the Class II Major Histocompatibility Complex Molecule That Leads to Cooperative Binding to a Superantigen

    SciTech Connect

    Li,H.; Zhao, Y.; Guo, Y.; Li, Z.; Eislele, L.; Mourad, W.

    2007-01-01

    Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor V{beta} elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn{sup 2+} is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn{sup 2+}. However, in the presence of Zn{sup 2+}, a dimerized MAM/HLA-DR1/HA complex can arise through the Zn{sup 2+}-induced DR1 dimer. In the presence of Zn{sup 2+}, cooperative binding of MAM to the DR1 dimer was also observed.

  4. Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses

    NASA Astrophysics Data System (ADS)

    Borgwardt, Derek S.; Martin, Aaron D.; van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.

    2014-01-01

    Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

  5. Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

    PubMed

    Zimmerman, Seth P; Hallett, Ryan A; Bourke, Ashley M; Bear, James E; Kennedy, Matthew J; Kuhlman, Brian

    2016-09-20

    Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

  6. Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

    PubMed

    Zimmerman, Seth P; Hallett, Ryan A; Bourke, Ashley M; Bear, James E; Kennedy, Matthew J; Kuhlman, Brian

    2016-09-20

    Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light. PMID:27529180

  7. Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation

    PubMed Central

    Bertucci, Paola Y.; Nacht, A. Silvina; Alló, Mariano; Rocha-Viegas, Luciana; Ballaré, Cecilia; Soronellas, Daniel; Castellano, Giancarlo; Zaurin, Roser; Kornblihtt, Alberto R.; Beato, Miguel; Vicent, Guillermo P.; Pecci, Adali

    2013-01-01

    Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3′-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes. PMID:23640331

  8. Calculated distortions induced by metal-ion binding to simple oligonucleotide systems: Implications for toxicity

    SciTech Connect

    Turner, J.E.; Hingerty, B.E.; England, M.W.; Jacobson, K.B.

    1990-01-01

    We have previously published detailed results of calculations of the binding of the metal ions, Cd{sup 2+} and Ca{sup 2+}, to the dinucleoside monophosphate GpC in water. These ions, which have the same charge and radius, differ enormously in their toxicity to man and other biological systems. Our calculations showed contrasting behavior in the binding of these two metal ions to GpC. We suggest the hypothesis that structural distortions calculated for metal ions binding to simple nucleic-acid systems might serve as a indicator of an ion's potential ability to alter molecular activity and hence to be toxic to an organism. Furthermore, the degree of distortion might be correlated with the degree of toxicity as measured by some suitable criteria. The present paper reports the results of binding calculations for a number of other metal ions, of different valence states, with several dinucleoside monophosphates in water. A general trend of distortion with the type of binding of the metal ions is found. We are seeking quantitative measures of distortion to correlate with indicators of acute toxicity that we have measured for 24 metal ions using mice, Drosophila, and CHO cells. 3 refs., 3 figs.

  9. CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

    PubMed

    Aguilar-Morante, Diana; Morales-Garcia, Jose A; Santos, Angel; Perez-Castillo, Ana

    2015-02-28

    We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells. PMID:25738360

  10. CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

    PubMed

    Aguilar-Morante, Diana; Morales-Garcia, Jose A; Santos, Angel; Perez-Castillo, Ana

    2015-02-28

    We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells.

  11. CCAAT/Enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4

    PubMed Central

    Aguilar-Morante, Diana; Morales-Garcia, Jose A.; Santos, Angel; Perez-Castillo, Ana

    2015-01-01

    We have previously shown that decreased expression of CCAAT/Enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells. PMID:25738360

  12. The metalloid arsenite induces nuclear export of Id3 possibly via binding to the N-terminal cysteine residues

    SciTech Connect

    Kurooka, Hisanori; Sugai, Manabu; Mori, Kentaro; Yokota, Yoshifumi

    2013-04-19

    Highlights: •Sodium arsenite induces cytoplasmic accumulation of Id3. •Arsenite binds to closely spaced N-terminal cysteine residues of Id3. •N-terminal cysteines are essential for arsenite-induced nuclear export of Id3. •Nuclear export of Id3 counteracts its transcriptional repression activity. -- Abstract: Ids are versatile transcriptional repressors that regulate cell proliferation and differentiation, and appropriate subcellular localization of the Id proteins is important for their functions. We previously identified distinct functional nuclear export signals (NESs) in Id1 and Id2, but no active NES has been reported in Id3. In this study, we found that treatment with the stress-inducing metalloid arsenite led to the accumulation of GFP-tagged Id3 in the cytoplasm. Cytoplasmic accumulation was impaired by a mutation in the Id3 NES-like sequence resembling the Id1 NES, located at the end of the HLH domain. It was also blocked by co-treatment with the CRM1-specific nuclear export inhibitor leptomycin B (LMB), but not with the inhibitors for mitogen-activated protein kinases (MAPKs). Importantly, we showed that the closely spaced N-terminal cysteine residues of Id3 interacted with the arsenic derivative phenylarsine oxide (PAO) and were essential for the arsenite-induced cytoplasmic accumulation, suggesting that arsenite induces the CRM1-dependent nuclear export of Id3 via binding to the N-terminal cysteines. Finally, we demonstrated that Id3 significantly repressed arsenite-stimulated transcription of the immediate-early gene Egr-1 and that this repression activity was inversely correlated with the arsenite-induced nuclear export. Our results imply that Id3 may be involved in the biological action of arsenite.

  13. Differential Complement Activation Pathways Promote C3b Deposition on Native and Acetylated LDL thereby Inducing Lipoprotein Binding to the Complement Receptor 1

    PubMed Central

    Klop, Boudewijn; van der Pol, Pieter; van Bruggen, Robin; Wang, Yanan; de Vries, Marijke A.; van Santen, Selvetta; O'Flynn, Joseph; van de Geijn, Gert-Jan M.; Njo, Tjin L.; Janssen, Hans W.; de Man, Peter; Jukema, J. Wouter; Rabelink, Ton J.; Rensen, Patrick C. N.; van Kooten, Cees; Cabezas, Manuel Castro

    2014-01-01

    Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1. PMID:25349208

  14. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

    PubMed Central

    Wang, Tao; Darwin, K. Heran; Li, Huilin

    2010-01-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analogue of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein bearing little sequence or structural resemblance to the highly structured ubiquitin. Thus it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled-coils that recognize Pup. Mpa binds unstructured Pup via hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work revealed a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This critical difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment of tuberculosis. PMID:20953180

  15. Exploring the capabilities of TDDFT calculations to explain the induced chirality upon a binding process: A simple case, 3-carboxycoumarin

    NASA Astrophysics Data System (ADS)

    Varlan, Aurica; Hillebrand, Mihaela

    2013-03-01

    The induced circular dichroism (ICD) spectra of 3-carboxycoumarin recorded at pH 7.4 in the presence of human and bovine serum albumins were used in correlation with theoretical (TDDFT) calculations to obtain the binding constants and information on the conformational changes of the ligand in the binding site. As it was shown that for the carboxylic acids or the carboxylate ions, the asymmetry element correlated with the occurrence of the ICD band in the presence of proteins is the torsion (τ) of the COOH (COO-) group in respect with the planar π system, TDDFT calculations were performed considering all the geometries characterized by 0 ⩽ |τ| ⩽ 90 deg. The simulated circular dichroism spectrum shows that the sequence of the signs and positions of the bands are correctly predicted as compared to the experimental ICD spectrum for a torsion of the carboxylate group in the range of 60-70 deg.

  16. Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA.

    PubMed

    Acevedo, Roderico; Evans, Declan; Penrod, Katheryn A; Showalter, Scott A

    2016-06-21

    Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.

  17. Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA.

    PubMed

    Acevedo, Roderico; Evans, Declan; Penrod, Katheryn A; Showalter, Scott A

    2016-06-21

    Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD's binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol(-1)·K(-1)) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed. PMID:27332119

  18. Direct observation of binding stress-induced crystalline orientation change in piezoelectric plate sensors

    NASA Astrophysics Data System (ADS)

    Wu, Wei; Shih, Wei-Heng; Shih, Wan Y.

    2016-03-01

    We have examined the mechanism of the detection resonance frequency shift, Δf/f, of a 1370 μm long and 537 μm wide [Pb(Mg1/3Nb2/3)O3]0.65[PbTiO3]0.35 (PMN-PT) piezoelectric plate sensor (PEPS) made of a 8-μm thick PMN-PT freestanding film. The Δf/f of the PEPS was monitored in a three-step binding model detections of (1) binding of maleimide-activated biotin to the sulfhydryl on the PEPS surface followed by (2) binding of streptavidin to the bound biotin and (3) subsequent binding of biotinylated probe deoxyribonucleic acid to the bound streptavidin. We used a PMN-PT surrogate made of the same 8-μm thick PMN-PT freestanding film that the PEPS was made of but was about 1 cm in length and width to carry out crystalline orientation study using X-ray diffraction (XRD) scan around the (002)/(200) peaks after each of the binding steps. The result of the XRD studies indicated that each binding step caused the crystalline orientation of the PMN-PT thin layer to switch from the vertical (002) orientation to the horizontal (200) orientation, and most of the PEPS detection Δf/f was due to the change in the lateral Young's modulus of the PMN-PT thin layer as a result of the crystalline orientation change.

  19. Cancer-associated S100P protein binds and inactivates p53, permits therapy-induced senescence and supports chemoresistance

    PubMed Central

    Gibadulinova, Adriana; Pastorek, Michal; Filipcik, Pavel; Radvak, Peter; Csaderova, Lucia; Vojtesek, Borivoj; Pastorekova, Silvia

    2016-01-01

    S100P belongs to the S100 family of calcium-binding proteins regulating diverse cellular processes. Certain S100 family members (S100A4 and S100B) are associated with cancer and used as biomarkers of metastatic phenotype. Also S100P is abnormally expressed in tumors and implicated in migration-invasion, survival, and response to therapy. Here we show that S100P binds the tumor suppressor protein p53 as well as its negative regulator HDM2, and that this interaction perturbs the p53-HDM2 binding and increases the p53 level. Paradoxically, the S100P-induced p53 is unable to activate its transcriptional targets hdm2, p21WAF, and bax following the DNA damage. This appears to be related to reduced phosphorylation of serine residues in both N-terminal and C-terminal regions of the p53 molecule. Furthermore, the S100P expression results in lower levels of pro-apoptotic proteins, in reduced cell death response to cytotoxic treatments, followed by stimulation of therapy-induced senescence and increased clonogenic survival. Conversely, the S100P silencing suppresses the ability of cancer cells to survive the DNA damage and form colonies. Thus, we propose that the oncogenic role of S100P involves binding and inactivation of p53, which leads to aberrant DNA damage responses linked with senescence and escape to proliferation. Thereby, the S100P protein may contribute to the outgrowth of aggressive tumor cells resistant to cytotoxic therapy and promote cancer progression. PMID:26967060

  20. Role of guanine nucleotide binding protein(s) in vasopressin-induced responses of a vascular smooth muscle cell line

    SciTech Connect

    Nambi, P.; Aiyar, N.; Whitman, M.; Stassen, F.L.; Crooke, S.T.

    1986-05-01

    Rat aortic smooth muscle cells (A-10) carry vascular V1 vasopressin receptors. In these cells, vasopressin inhibits isoproterenol-induced cAMP accumulation and stimulates phosphatidylinositol turnover and Ca/sup 2 +/ mobilization. Pretreatment of the cells with phorbol esters resulted in inhibition of the vasopressin-induced responses. The inactive phorbol ester aPDD was ineffective. These data suggested that phorbol ester might cause phosphorylation of the vasopressin receptor and/or coupling protein(s). Here, they studied the role of guanine nucleotide binding proteins by employing the novel radiolabeled vasopressin antagonist (/sup 3/H)-SKF 101926. In competition experiments with cell membranes, Gpp(NH)p shifted the vasopressin curve to the right indicating decreased agonist affinity. Phorbol ester pretreatment abolished the Gpp(NH)p effect. Pretreatment of the cells with N-ethylmaleimide (NEM) resulted in inhibition of vasopressin-induced phosphatidyinositol turnover. NEM also abolished the decrease in agonist affinity caused by Gpp(NH)p. These data showed that NEM and phorbol ester pretreatment of smooth muscle cells functionally uncoupled the vasopressin receptors and suggested that vasopressin V1 receptor responses are mediated through guanine nucleotide binding protein(s).

  1. Heat-induced alterations in cashew allergen solubility and IgE binding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cashew nuts are included in a group of 8 foods that commonly cause food allergies. IgE binding to allergens within the nuts can cause allergic reactions that can be severe. Foods containing cashew nuts must be labeled to prevent accidental exposure to people who suffer from allergy to cashew nuts....

  2. Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding.

    PubMed

    Bullen, Gemma A; Tucker, James H R; Peacock, Anna F A

    2015-05-11

    The unprecedented use of anthracene photodimerization within a protein or peptide system is explored through its incorporation into a DNA-binding peptide, derived from the GCN4 transcription factor. This study demonstrates an effective and dynamic interplay between a photoreaction and a peptide-DNA assembly, with each process able to exert control over the other.

  3. Structures of the inducer-binding domain of pentachlorophenol-degrading gene regulator PcpR from Sphingobium chlorophenolicum.

    PubMed

    Hayes, Robert P; Moural, Timothy W; Lewis, Kevin M; Onofrei, David; Xun, Luying; Kang, ChulHee

    2014-01-01

    PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 μM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol. PMID:25397598

  4. Human natural immunoglobulin M antibodies induce apoptosis of human neuroblastoma cells by binding to a Mr 260,000 antigen.

    PubMed

    David, K; Ollert, M W; Vollmert, C; Heiligtag, S; Eickhoff, B; Erttmann, R; Bredehorst, R; Vogel, C W

    1999-08-01

    Sera of healthy humans contain natural cytotoxic IgM antibodies that specifically recognize a Mr 260,000 antigen (NB-p260) on the surface of human neuroblastoma (NB) cells. Here we demonstrate that anti-NB IgM antibodies prepared from different healthy individuals induce, in all human NB cell lines analyzed thus far, typical morphological and biochemical features of apoptosis including nuclear fragmentation, chromatin condensation, and DNA fragmentation. Both the binding of human anti-NB IgM to NB cells and the induction of apoptosis could be inhibited by preincubation of NB cells with murine IgG raised against purified NB-p260. Furthermore, preincubation of human anti-NB IgM with purified NB-p260 immobilized onto a solid support abolished its ability to induce apoptosis in NB cells. Natural human anti-NB IgM failed to bind to and induce apoptosis in control tumor cell lines that lack expression of NB-p260. The anti-NB IgM-induced apoptotic response was also observed in vivo in xenografted human NB tumors. After a single i.v. injection of anti-NB IgM into nude rats bearing solid NB xenografts, many areas of pyknotic cells with fragmented nuclei were observed that stained positive using the terminal dUTP nick end labeling method. In conclusion, the data demonstrate that natural anti-NB IgM antibodies in the sera of healthy individuals are potent mediators of apoptotic cell death of NB cells both in vitro and in vivo. The NB-p260 antigen was identified as the apoptosis-inducing receptor for anti-NB IgM. Whereas natural anti-NB IgM and NB-p260 may be useful tools for immunotherapy of NB, their biological significance remains to be determined.

  5. Role of inducer binding in cytochrome P-450 IA2-mediated uroporphyrinogen oxidation.

    PubMed

    Jacobs, J M; Sinclair, P R; Lambrecht, R W; Sinclair, J F; Jacobs, N J

    1990-01-01

    The oxidation of uroporphyrinogen, an intermediate of the heme biosynthetic pathway, by methylcholanthrene-inducible isozymes(s) of cytochrome P-450 has been proposed to play a role in the development of chemically induced uroporphyria. Prior work from this laboratory indicated that although addition of 3,4,3',4'-tetrachlorobiphenyl is required for uroporphyrinogen oxidation by methylcholanthrene-induced chick embryo liver microsomes, this biphenyl is not required for the oxidation catalyzed by hepatic microsomes from methylcholanthrene-induced rodents. Here we investigated whether rodent microsomes catalyze uroporphyrinogen oxidation without addition of 3,4,3',4'-tetrachlorobiphenyl because the chemical used as an inducer remains bound to cytochrome P-450. Hepatic microsomes containing almost no residual inducer were isolated from rats treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These microsomes oxidized uroporphyrinogen at high rates without addition of 3,4,3',4'-tetrachlorobiphenyl. Inducer-free microsomal cytochrome P-450 was also obtained by inducing cytochrome P-450 in rats and mice with isosafrole, which was then removed from the isolated microsomes by butanol treatment. This procedure resulted in microsomes with high activity for uroporphyrinogen oxidation. Furthermore, addition of chlorobiphenyl to these inducer-free microsomes was inhibitory. Hepatic microsomes from isosafrole-induced C57BL/6 and DBA mice, rendered inducer-free by butanol treatment, oxidized uroporphyrinogen at the same rate even though these two strains differ markedly in their susceptibility to chemically induced uroporphyria. We conclude that uroporphyrinogen oxidation is catalyzed by cytochrome P-450 that is free of inducer.

  6. Anion-induced increases in the rate of colchicine binding to tubulin.

    PubMed

    Bhattacharyya, B; Wolff, J

    1976-06-01

    The rate of binding of colchicine to tubulin to tubulin is enhanced by certain anions. Among the inorganic anions tested, only sulfate was effective. The organic anions include mostly dicarboxylic acids, among which tartrate was the most effective. This effect occurs onlt at low concentrations of colchicine (less than 0.6 X 10(-5) M). The rate increase dor sulfate and L-(+)-tartrate is ca. 2.5-fold at 1.0 mM and plateaus at a limiting value of ca. 4-fold at 100mM. The overall dissociation rate of the colchicine from the complex, which includes both the true rate of dissociation and the rate of irreversible denaturation of tubulin, is not influenced by 1.0 mM tartrate. The affinity constants for colchicine determined from the rate constants are 8.7 X 10(6) and 2.1 X 10(7) M-1 in the absence and the presence of 1.0 mM L-(+)-tartrate. The limiting value is 3.2 X 10(7) M-1. The affinity constant calculated from steady-state measurements is 3.2 X 10(6) M-1 with or without anions. The binding of other ligands like podophyllotoxin, vinblastine, and 1 -anilino-8-naphthalenesulfonate to tubulin is not affected by tartrate. No major conformational changes resulting from anion treatment could be detected by circular dichroism or intrinsic fluorescence. However, the ability of tubulin to polymerize is inhibited by L-(+)-tartrate at concentrations that increase the rate of colchicine binding. We conclude that anions must have a local effect at or near the binding site which enhances the binding rate of colchicine and which may be related to inhibition of polymerization.

  7. Ezrin-radixin-moesin-binding phosphoprotein-50 regulates EGF-induced AKT activation through interaction with EGFR and PTEN.

    PubMed

    Zheng, Junfang; Dai, Yuanping; Yang, Zhiyu; Yang, Longyan; Peng, Zhiqiang; Meng, Ran; Xiong, Ying; He, Junqi

    2016-01-01

    Dysregulated epidermal growth factor receptor (EGFR) signaling, especially EGFR/AKT signaling, plays important roles in tumorigenesis and progression, the study on intracellular regulation of this signaling pathway has great clinical significance. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important antagonist of AKT activity. Its regulation of AKT activity can be enhanced by ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50)-mediated PTEN/EBP50/platelet-derived growth factor receptor (PDGFR) complex. EBP50 was reported to bind to EGFR, and that it may also mediate the formation of PTEN/EGFR complex to regulate EGFR/AKT signaling. In this study, experiments were performed to verify the hypothesis. Results showed that PTEN co-immunoprecipitated with EGFR, demonstrating PTEN/EGFR complex can form in tissue. Further studies showed that EBP50 knockdown decreased the amount of PTEN/EGFR complex by GST pull-down assay, and EBP50 overexpression increased the amount of PTEN/EGFR complex in a dose-dependent manner. While PTEN mutant (V403A), which can not bind with EBP50, only slightly mediated the formation of PTEN/EGFR complex, confirming that EBP50 specifically mediated the formation of the PTEN/EGFR complex. Both PTEN (V403A) and EGFR (L1043/1063F) mutants can not bind with EBP50. The expression of PTEN (V403A) or EGFR (L1043/1063F) mutant in cells resulted in higher AKT activation level than their respective wild-types by EGF stimulation, indicating that EBP50-mediated PTEN/EGFR complex can effectively inhibit EGF-induced AKT activation. EGF stimulation of siEBP50 cells induced higher AKT activation level compared with control cells, further confirming EBP50-mediated PTEN/EGFR complex can more effectively inhibit EGF-induced AKT activation. These results demonstrated the PTEN/EGFR complex formed under the mediation of EBP50, revealing a novel mechanism for negative regulation of EGF-induced AKT pathway, which may be an important molecular

  8. GRAS Proteins Form a DNA Binding Complex to Induce Gene Expression during Nodulation Signaling in Medicago truncatula[W

    PubMed Central

    Hirsch, Sibylle; Kim, Jiyoung; Muñoz, Alfonso; Heckmann, Anne B.; Downie, J. Allan; Oldroyd, Giles E.D.

    2009-01-01

    The symbiotic association of legumes with rhizobia involves bacterially derived Nod factor, which is sufficient to activate the formation of nodules on the roots of the host plant. Perception of Nod factor by root hair cells induces calcium oscillations that are a component of the Nod factor signal transduction pathway. Perception of the calcium oscillations is a function of a calcium- and calmodulin-dependent protein kinase, and this activates nodulation gene expression via two GRAS domain transcriptional regulators, Nodulation Signaling Pathway1 (NSP1) and NSP2, and an ERF transcription factor required for nodulation. Here, we show that NSP1 and NSP2 form a complex that is associated with the promoters of early nodulin genes. We show that NSP1 binds directly to ENOD promoters through the novel cis-element AATTT. While NSP1 shows direct binding to the ENOD11 promoter in vitro, this association in vivo requires NSP2. The NSP1-NSP2 association with the ENOD11 promoter is enhanced following Nod factor elicitation. Mutations in the domain of NSP2 responsible for its interaction with NSP1 highlight the significance of the NSP1-NSP2 heteropolymer for nodulation signaling. Our work reveals direct binding of a GRAS protein complex to DNA and highlights the importance of the NSP1-NSP2 complex for efficient nodulation in the model legume Medicago truncatula. PMID:19252081

  9. Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

    PubMed

    Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F

    2016-01-01

    Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

  10. Plasminogen activator inhibitor 1--insulin-like growth factor binding protein 3 cascade regulates stress-induced senescence.

    PubMed

    Elzi, David J; Lai, Yanlai; Song, Meihua; Hakala, Kevin; Weintraub, Susan T; Shiio, Yuzuru

    2012-07-24

    Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescence-inducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.

  11. Manganese ions induce H2O2 generation at the ubiquinone binding site of mitochondrial complex II.

    PubMed

    Bonke, Erik; Zwicker, Klaus; Dröse, Stefan

    2015-08-15

    Manganese-induced toxicity has been recently associated with an increased ROS generation from mitochondrial complex II (succinate:ubiquinone oxidoreductase). To achieve a deeper mechanistic understanding how divalent manganese ions (Mn(2+)) could stimulate mitochondrial ROS production we performed investigations with bovine heart submitochondrial particles (SMP). In succinate fueled SMP, the Mn(2+) induced hydrogen peroxide (H2O2) production was blocked by the specific complex II ubiquinone binding site (IIQ) inhibitor atpenin A5 while a further downstream block at complex III increased the rate markedly. This suggests that site IIQ was the source of the reactive oxygen species. Moreover, Mn(2+) ions also accelerated the rate of superoxide dismutation, explaining the general increase in the measured rates of H2O2 production and an attenuation of direct superoxide detection.

  12. Induced conformational change in human IL‐4 upon binding of a signal‐neutralizing DARPin

    PubMed Central

    Teplyakov, Alexey; Malia, Thomas J.; Keough, Edward; Luo, Jinquan; Sweet, Raymond; Jacobs, Steven A.; Yi, Fang; Hippensteel, Randi; O'Neil, Karyn T.

    2015-01-01

    ABSTRACT The crystal structure of DARPin 44C12V5 that neutralizes IL‐4 signaling has been determined alone and bound to human IL‐4. A significant conformational change occurs in the IL‐4 upon DARPin binding. The DARPin binds to the face of IL‐4 formed by the A and C α‐helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL‐4 include a reorientation of the C‐helix Trp91 side chain and repositioning of CD‐loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL‐4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical “hotspot.” The structures also reveal how the DARPin neutralizes IL‐4 signaling. Comparing the IL‐4:DARPin complex structure with the structures of IL‐4 bound to its receptors (Hage et al., Cell 1999; 97, 271‐281; La Porte et al., Cell 2008, 132, 259‐272), it is found that the DARPin binds to the same IL‐4 face that interacts with the junction of the D1 and D2 domains of the IL‐4Rα receptors. Signaling is blocked since IL‐4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL‐13α1 or the γ c receptor. Proteins 2015; 83:1191–1197. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25900776

  13. Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase.

    PubMed

    Salinas, S R; Petruk, A A; Brukman, N G; Bianco, M I; Jacobs, M; Marti, M A; Ielpi, L

    2016-06-01

    GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site. PMID:27099353

  14. Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins.

    PubMed

    Vincent, S H; Holeman, B; Cully, B C; Muller-Eberhard, U

    1986-01-27

    Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

  15. Functional and structural changes of human erythrocyte catalase induced by cimetidine: proposed model of binding.

    PubMed

    Yazdi, Fatemeh; Minai-Tehrani, Dariush; Jahngirvand, Mahboubeh; Almasirad, Ali; Mousavi, Zahra; Masoud, Masoudeh; Mollasalehi, Hamidreza

    2015-06-01

    In erythrocyte, catalase plays an important role to protect cells from hydrogen peroxide toxicity. Hydrogen peroxide is a byproduct compound which is produced during metabolic pathway of cells. Cimetidine, a histamine H2 receptor antagonist, is used for gastrointestinal tract diseases and prevents the extra release of gastric acid. In this study, the effect of cimetidine on the activity of human erythrocyte catalase was investigated. Erythrocytes were broken by hypotonic solution. The supernatant was used for catalase assay and kinetics study. Lineweaver-Burk plot was performed to determine the type of inhibition. The kinetics data revealed that cimetidine inhibited the catalase activity by mixed inhibition. The IC50 (1.54 μM) and Ki (0.45 μM) values of cimetidine determined that the drug was bound to the enzyme with high affinity. Circular dichroism and fluorescence measurement showed that the binding of cimetidine to the enzyme affected the content of secondary structure of the enzyme as well as its conformational changes. Docking studies were carried out to detect the site in which the drug was bound to the enzyme. Molecular modeling and energy calculation of the binding showed that the cyanoguanidine group of the drug connected to Asp59 via two hydrogen bonds, while the imidazole group of the drug interacted with Phe64 in the enzyme by a hydrophobic interaction. In conclusion, cimetidine could bind to human erythrocyte catalase, and its interaction caused functional and conformational changes in the enzyme.

  16. Binding of Folic Acid Induces Specific Self-Aggregation of Lactoferrin: Thermodynamic Characterization.

    PubMed

    Tavares, Guilherme M; Croguennec, Thomas; Lê, Sébastien; Lerideau, Olivia; Hamon, Pascaline; Carvalho, Antônio F; Bouhallab, Saïd

    2015-11-17

    In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks. PMID:26488446

  17. Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

    PubMed Central

    Haq, Tamanna; Richards, Mark W.; Burgess, Selena G.; Gallego, Pablo; Yeoh, Sharon; O'Regan, Laura; Reverter, David; Roig, Joan; Fry, Andrew M.; Bayliss, Richard

    2015-01-01

    Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810–828. A crystal structure of Nek7Y97F bound to Nek9810–828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families. PMID:26522158

  18. Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization

    NASA Astrophysics Data System (ADS)

    Haq, Tamanna; Richards, Mark W.; Burgess, Selena G.; Gallego, Pablo; Yeoh, Sharon; O'Regan, Laura; Reverter, David; Roig, Joan; Fry, Andrew M.; Bayliss, Richard

    2015-11-01

    Mitotic spindle assembly requires the regulated activities of protein kinases such as Nek7 and Nek9. Nek7 is autoinhibited by the protrusion of Tyr97 into the active site and activated by the Nek9 non-catalytic C-terminal domain (CTD). CTD binding apparently releases autoinhibition because mutation of Tyr97 to phenylalanine increases Nek7 activity independently of Nek9. Here we find that self-association of the Nek9-CTD is needed for Nek7 activation. We map the minimal Nek7 binding region of Nek9 to residues 810-828. A crystal structure of Nek7Y97F bound to Nek9810-828 reveals a binding site on the C-lobe of the Nek7 kinase domain. Nek7Y97F crystallizes as a back-to-back dimer between kinase domain N-lobes, in which the specific contacts within the interface are coupled to the conformation of residue 97. Hence, we propose that the Nek9-CTD activates Nek7 through promoting back-to-back dimerization that releases the autoinhibitory tyrosine residue, a mechanism conserved in unrelated kinase families.

  19. Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

    PubMed

    Pai, Jaeyoung; Hyun, Soonsil; Hyun, Ji Young; Park, Seong-Hyun; Kim, Won-Je; Bae, Sung-Hun; Kim, Nak-Kyoon; Yu, Jaehoon; Shin, Injae

    2016-01-27

    MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing. PMID:26771315

  20. OSTEOPONTIN BINDING TO LIPOPOLYSACCHARIDE LOWERS TUMOR NECROSIS FACTOR-α AND PREVENTS EARLY ALCOHOL-INDUCED LIVER INJURY IN MICE

    PubMed Central

    Ge, Xiaodong; Leung, Tung-Ming; Arriazu, Elena; Lu, Yongke; Urtasun, Raquel; Christensen, Brian; Fiel, Maria Isabel; Mochida, Satoshi; Sørensen, Esben S.; Nieto, Natalia

    2013-01-01

    Rationale: Although osteopontin (OPN) is induced in alcoholic patients, its role in the pathophysiology of alcoholic liver disease (ALD) remains unclear. Increased translocation of lipopolysaccharide (LPS) from the gut is key for the onset of ALD since it promotes macrophage infiltration and activation, tumor necrosis factor-α (TNFα) production and liver injury. Since OPN is protective for the intestinal mucosa, we postulated that enhancing OPN expression in the liver and consequently in the blood and/or in the gut could protect from early alcohol-induced liver injury. Results: Wild-type (WT), OPN knockout (Opn−/−) and transgenic mice overexpressing OPN in hepatocytes (OpnHEP Tg) were chronically fed either the control or the ethanol Lieber-DeCarli diet. Ethanol increased hepatic, plasma, biliary and fecal OPN more in OpnHEP Tg than in WT mice. Steatosis was lesser in ethanol-treated OpnHEP Tg mice as shown by decreased liver-to-body weight ratio, hepatic triglycerides, the steatosis score, oil red-O staining and lipid peroxidation. There was also less inflammation and liver injury as demonstrated by lower ALT activity, hepatocyte ballooning degeneration, LPS levels, the inflammation score and the number of macrophages and TNFα+ cells. To establish if OPN could limit LPS availability and its noxious effects in the liver, binding studies were performed. OPN showed affinity for LPS and the binding prevented macrophage activation, reactive oxygen and nitrogen species generation and TNFα production. Treatment with milk OPN (m-OPN) blocked LPS translocation in vivo and protected from early alcohol-induced liver injury. Conclusion: Natural induction plus forced overexpression of OPN in the liver and treatment with m-OPN protect from early alcohol-induced liver injury by blocking the gut-derived LPS and TNFα effects in the liver. PMID:24214181

  1. Characterization of an Acidic-pH-Inducible Stress Protein (hsp70), a Putative Sulfatide Binding Adhesin, from Helicobacter pylori

    PubMed Central

    Huesca, Mario; Goodwin, Avery; Bhagwansingh, Arianna; Hoffman, Paul; Lingwood, Clifford A.

    1998-01-01

    The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress. PMID:9712748

  2. Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2☆

    PubMed Central

    Shimahara, Hideto; Hirano, Takaaki; Ohya, Kouichi; Matsuta, Shun; Seeram, Sailaja S.; Tate, Shin-ichi

    2013-01-01

    Interactions between the nucleosome and the non-histone chromosomal proteins (HMGN1 and HMGN2) were studied by circular dichroism (CD) spectroscopy to elucidate structural changes in the nucleosome induced by HMGN binding. Unlike previous studies that used a nucleosome extracted from living cells, in this study we utilized a nucleosome reconstituted from unmodified recombinant histones synthesized in Escherichia coli and a 189-bp synthetic DNA fragment harboring a nucleosome positioning sequence. This DNA fragment consists of 5′-TATAAACGCC-3′ repeats that has a high affinity to the histone octamer. A nucleosome containing a unique octamer-binding sequence at a specific location on the DNA was produced at sufficiently high yield for spectroscopic analysis. CD data have indicated that both HMGN1 and HMGN2 can increase the winding angle of the nucleosome DNA, but the extent of the structural changes induced by these proteins differs significantly. This suggests HMGN1 and HMGN2 would have different abilities to facilitate nucleosome remodeling. PMID:23772392

  3. A galactose-binding lectin isolated from Aplysia kurodai (sea hare) eggs inhibits streptolysin-induced hemolysis.

    PubMed

    Hasan, Imtiaj; Watanabe, Miharu; Ishizaki, Naoto; Sugita-Konishi, Yoshiko; Kawakami, Yasushi; Suzuki, Jun; Dogasaki, Chikaku; Rajia, Sultana; Kawsar, Sarkar M A; Koide, Yasuhiro; Kanaly, Robert A; Sugawara, Shigeki; Hosono, Masahiro; Ogawa, Yukiko; Fujii, Yuki; Iriko, Hideyuki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2014-01-01

    A specific galactose-binding lectin was shown to inhibit the hemolytic effect of streptolysin O (SLO), an exotoxin produced by Streptococcus pyogenes. Commercially available lectins that recognize N-acetyllactosamine (ECA), T-antigen (PNA), and Tn-antigen (ABA) agglutinated rabbit erythrocytes, but had no effect on SLO-induced hemolysis. In contrast, SLO-induced hemolysis was inhibited by AKL, a lectin purified from sea hare (Aplysia kurodai) eggs that recognizes α-galactoside oligosaccharides. This inhibitory effect was blocked by the co-presence of d-galactose, which binds to AKL. A possible explanation for these findings is that cholesterol-enriched microdomains containing glycosphingolipids in the erythrocyte membrane become occupied by tightly stacked lectin molecules, blocking the interaction between cholesterol and SLO that would otherwise result in penetration of the membrane. Growth of S. pyogenes was inhibited by lectins from a marine invertebrate (AKL) and a mushroom (ABA), but was promoted by a plant lectin (ECA). Both these inhibitory and promoting effects were blocked by co-presence of galactose in the culture medium. Our findings demonstrate the importance of glycans and lectins in regulating mechanisms of toxicity, creation of pores in the target cell membrane, and bacterial growth. PMID:25197935

  4. Pheromone-induced morphogenesis and gradient tracking are dependent on the MAPK Fus3 binding to Gα

    PubMed Central

    Errede, Beverly; Vered, Lior; Ford, Eintou; Pena, Matthew I.; Elston, Timothy C.

    2015-01-01

    Mitogen-activated protein kinase (MAPK) pathways control many cellular processes, including differentiation and proliferation. These pathways commonly activate MAPK isoforms that have redundant or overlapping function. However, recent studies have revealed circumstances in which MAPK isoforms have specialized, nonoverlapping roles in differentiation. The mechanisms that underlie this specialization are not well understood. To address this question, we sought to establish regulatory mechanisms that are unique to the MAPK Fus3 in pheromone-induced mating and chemotropic fate transitions of the budding yeast Saccharomyces cerevisiae. Our investigations reveal a previously unappreciated role for inactive Fus3 as a potent negative regulator of pheromone-induced chemotropism. We show that this inhibitory role is dependent on inactive Fus3 binding to the α-subunit of the heterotrimeric G-protein. Further analysis revealed that the binding of catalytically active Fus3 to the G-protein is required for gradient tracking and serves to suppress cell-to-cell variability between mating and chemotropic fates in a population of pheromone-responding cells. PMID:26179918

  5. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    PubMed Central

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  6. Acetylation of the proto-oncogene EVI1 abrogates Bcl-xL promoter binding and induces apoptosis.

    PubMed

    Pradhan, Anjan Kumar; Mohapatra, Alok Das; Nayak, Kasturi Bala; Chakraborty, Soumen

    2011-01-01

    EVI1 (Ecotropic Viral Integration site I), which was originally identified as a myeloid transforming gene by means of retroviral insertional mutagenesis in mouse leukemia, encodes a nuclear DNA binding zinc finger protein. The presence of zinc fingers that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, except a few, target genes of EVI1 are poorly functionally identified thus far. In this study we provide evidence that EVI1 directly induces the expression of Bcl-xL through the first set of zinc finger and thereby inhibits apoptosis. ChIP analysis showed that EVI1 binds to the Bcl-xL promoter in HT-29 cells, a colon carcinoma cell line, which expresses EVI1. The observation is also supported by the fact that EVI1 siRNA treated HT-29 cells, shows a down regulation of Bcl-xL expression and that over expression of EVI1 results in the induction of the Bcl-xL reporter construct. A set of EVI1 positive chronic myeloid leukemia (CML) samples also showed higher Bcl-xL expression with respect to EVI1 negative samples. Interestingly, co-expression of EVI1 with wild type, but not with dominant-negative form of PCAF, abolishes the effect of EVI1 on Bcl-xL, indicating that acetylation of EVI1 abrogates its ability not only to bind Bcl-xL promoter but also alleviate Bcl-xL activity. Finally we have shown that EVI1 expression regulates apoptosis in HT-29 cells, which is abrogated when HT-29 cells are transfected with EVI1 siRNA or PCAF. The result for the first time shows a direct pathway by which EVI1 can protect cells from apoptosis and also demonstrates that the pathway can be reversed when EVI1 is acetylated.

  7. Inhibition of x-box binding protein 1 reduces tunicamycin-induced apoptosis in aged murine macrophages.

    PubMed

    Song, Yang; Shen, Hua; Du, Wei; Goldstein, Daniel R

    2013-10-01

    Endoplasmic reticulum (ER) stress is induced by the accumulation of unfolded and misfolded proteins in the ER. Although apoptosis induced by ER stress has been implicated in several aging-associated diseases, such as atherosclerosis, it is unclear how aging modifies ER stress response in macrophages. To decipher this relationship, we assessed apoptosis in macrophages isolated from young (1.5-2 months) and aged (16-18 months) mice and exposed the cells to the ER stress inducer tunicamycin. We found that aged macrophages exhibited more apoptosis than young macrophages, which was accompanied by reduced activation of phosphorylated inositol-requiring enzyme-1 (p-IRE1α), one of the three key ER stress signal transducers. Reduced gene expression of x-box binding protein 1 (XBP1), a downstream effector of IRE1α, enhanced p-IRE1α levels and reduced apoptosis in aged, but not young macrophages treated with tunicamycin. These findings delineate a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggest an important protective role of IRE1α in aging-associated ER stress-induced apoptosis. This novel pathway may not only be important in our understanding of longevity, but may also have important implications for pathogenesis and potential treatment of aging-associated diseases in general.

  8. Tight-binding model study of substrate induced pseudo-spin polarization and magnetism in mono-layer graphene

    NASA Astrophysics Data System (ADS)

    Sahu, Sivabrata; Rout, G. C.

    2016-06-01

    We present here a tight-binding model study of generation of magnetism and pseudo-spin polarization in monolayer graphene arising due to substrate, impurity and Coulomb correlation effects. The model Hamiltonian contains the first-, second- and third-nearest-neighbor hopping integrals for π electrons of graphene besides substrate induced gap, impurity interactions and Coulomb correlation of electrons. The Hubbard type Coulomb interactions present in both the sub-lattices A and B are treated within the mean-field approximation. The electronic Green's functions are calculated by using Zubarev's technique and hence the electron occupancies of both sub-lattices are calculated for up and down spins separately. These four temperature dependent occupancies are calculated numerically and self-consistently. Then we have calculated the temperature dependent pseudo-spin polarization, ferromagnetic and anti-ferromagnetic magnetizations. We observe that there exists pseudo-spin polarization for lower Coulomb energy, u < 2.2t1 and pseudo-spin polarization is enhanced with substrate induced gap and impurity effect. For larger Coulomb energy u > 2.5t1, there exists pseudo-spin polarization (p); while ferromagnetic (m) and antiferromagnetic (pm) magnetizations exhibit oscillatory behavior. With increase of the substrate induced gap, the ferromagnetic and antiferromagnetic transition temperatures are enhanced with increase of the substrate induced gap; while polarization (p) is enhanced in magnitude only.

  9. The ATP-Binding Cassette Transporter ABCB6 Is Induced by Arsenic and Protects against Arsenic Cytotoxicity

    PubMed Central

    Chavan, Hemantkumar; Oruganti, Mahitha; Krishnamurthy, Partha

    2011-01-01

    Arsenic, an environmental carcinogen, remains a major public health problem. Arsenic damages biological systems through multiple mechanisms, including the generation of reactive oxygen species. ABCB6 is an ATP-binding cassette transporter that is highly expressed in cells resistant to arsenic. We have recently demonstrated that ABCB6 expression protects against cellular stressors. In the present study, we evaluated the significance of ABCB6 expression to arsenic toxicity both in mice and in cell culture. We show that sodium arsenite induces ABCB6 expression in a dose-dependent manner both in mice fed sodium arsenite in drinking water and in cells exposed to sodium arsenite in vitro. Arsenite-induced ABCB6 expression was transcriptionally regulated, but this induction was not mediated by the redox-sensitive transcription factor nuclear factor-erythroid 2–related factor 2 (Nrf2). We demonstrate that, in HepG2 and Hep3B cells, knockdown of ABCB6 expression using ABCB6-specific small interfering RNA sensitized the cells to arsenite toxicity. In contrast, stable overexpression of ABCB6 conferred a strong survival advantage toward arsenite-induced oxidative stress. Collectively, these results, obtained by both loss of function and gain of function analysis, suggest that ABCB6 expression in response to sodium arsenite might be an endogenous protective mechanism activated to protect cells against arsenite-induced oxidative stress. PMID:21266531

  10. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    PubMed

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  11. Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding.

    PubMed

    Maas, Matthias; Wang, Ronggang; Paddock, Cathy; Kotamraju, Srigiridhar; Kalyanaraman, Balaraman; Newman, Peter J; Newman, Debra K

    2003-12-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) functions to control the activation and survival of the cells on which it is expressed. Many of the regulatory functions of PECAM-1 are dependent on its tyrosine phosphorylation and subsequent recruitment of the Src homology (SH2) domain containing protein tyrosine phosphatase SHP-2. The recent demonstration that PECAM-1 tyrosine phosphorylation occurs in cells exposed to the reactive oxygen species hydrogen peroxide (H2O2) suggested that this form of oxidative stress may also support PECAM-1/SHP-2 complex formation. In the present study, we show that PECAM-1 tyrosine phosphorylation in response to exposure of cells to H2O2 is reversible, involves a shift in the balance between kinase and phosphatase activities, and supports binding of SHP-2 and recruitment of this phosphatase to cell-cell borders. We speculate, however, that the unique ability of H2O2 to reversibly oxidize the reactive site cysteine residues of protein tyrosine phosphatases may result in transient inactivation of the SHP-2 that is bound to PECAM-1 under these conditions. Finally, we provide evidence that PECAM-1 tyrosine phosphorylation and SHP-2 binding in endothelial cells requires exposure to an "oxidative burst" of H2O2, but that exposure of these cells to sufficiently high concentrations of H2O2 for a sufficiently long period of time abrogates binding of SHP-2 to tyrosine-phosphorylated PECAM-1. These findings support a role for PECAM-1 as a sensor of oxidative stress, perhaps most importantly during the process of inflammation. PMID:12893640

  12. In silico analysis of molecular mechanisms of Galanthus nivalis agglutinin-related lectin-induced cancer cell death from carbohydrate-binding motif evolution hypothesis.

    PubMed

    Yu, Qi-Jia; Li, Zi-Yue; Yao, Shun; Ming, Miao; Wang, Shu-Ya; Liu, Bo; Bao, Jin-Ku

    2011-10-01

    Galanthus nivalis agglutinin-related lectins, a superfamily of strictly mannose-binding-specific lectins widespread amongst monotyledonous plants, have drawn a rising attention for their remarkable anti-proliferative and apoptosis-inducing activities toward various types of cancer cells; however, the precise molecular mechanisms by which they induce tumor cell apoptosis are still only rudimentarily understood. Herein, we found that the three conserved motifs "QXDXNXVXY," the mannose-specific binding sites, could mutate at one or more amino acid sites, which might be a driving force for the sequential evolution and thus ultimately leading to the complete disappearance of the three conserved motifs. In addition, we found that the motif evolution could result in the diversification of sugar-binding types that G. nivalis agglutinin-related lectins could bind from specific mannose receptors to more types of sugar-containing receptors in cancer cells. Subsequently, we indicated that some sugar-containing receptors such as TNFR1, EGFR, Hsp90, and Hsp70 could block downstream anti-apoptotic or survival signaling pathways, which, in turn, resulted in tumor cell apoptosis. Taken together, our hypothesis that carbohydrate-binding motif evolution may impact the G. nivalis agglutinin-related lectin-induced survival or anti-apoptotic pathways would provide a new perspective for further elucidating the intricate relationships between the carbohydrate-binding specificities and complex molecular mechanisms by which G. nivalis agglutinin-related lectins induce cancer cell death.

  13. The Panitumumab EGFR Complex Reveals a Binding Mechanism That Overcomes Cetuximab Induced Resistance

    PubMed Central

    Kurzeja, Robert J. M.; Michelsen, Klaus; Vazir, Mukta; Yang, Evelyn; Tasker, Andrew S.

    2016-01-01

    Panitumumab and cetuximab target the epidermal growth factor receptor for the treatment of metastatic colorectal cancer. These therapies provide a significant survival benefit to patients with metastatic colorectal cancer with wild-type RAS. A single point mutation in the ectodomain of EGFR (S468R) confers acquired or secondary resistance in cetuximab treated patients, which is not observed in panitumumab-treated patients. Structural and biophysical studies presented here show this mutation directly blocks cetuximab binding to EGFR domain III and describes a unique mechanism by which panitumumab uses a central cavity to accommodate this mutation. PMID:27658254

  14. Sub-inhibitory tigecycline concentrations induce extracellular matrix binding protein Embp dependent Staphylococcus epidermidis biofilm formation and immune evasion.

    PubMed

    Weiser, Julian; Henke, Hanae A; Hector, Nina; Both, Anna; Christner, Martin; Büttner, Henning; Kaplan, Jeffery B; Rohde, Holger

    2016-09-01

    Biofilm-associated Staphylococcus epidermidis implant infections are notoriously reluctant to antibiotic treatment. Here we studied the effect of sub-inhibitory concentrations of penicillin, oxacillin, vancomycin, daptomycin, linezolid and tigecycline on S. epidermidis 1585 biofilm formation, expression of extracellular matrix binding protein (Embp) and potential implications for S. epidermidis - macrophage interactions. Penicillin, vancomycin, daptomycin, and linezolid had no biofilm augmenting effect at any of the concentrations tested. In contrast, at sub-inhibitory concentrations tigecycline and oxacillin exhibited significant biofilm inducing activity. In S. epidermidis 1585, SarA is a negative regulator of giant 1 MDa Embp, and down regulation of sarA induces Embp-dependent assembly of a multi-layered biofilm architecture. Dot blot immune assays, confocal laser scanning microscopy, and qPCR showed that under biofilm inducing conditions, tigecycline augmented embp expression compared to the control grown without antibiotics. Conversely, expression of regulator sarA was suppressed, suggesting that tigecycline exerts its effects on embp expression through SarA. Tigecycline failed to induce biofilm formation in embp transposon mutant 1585-M135, proving that under these conditions Embp up-regulation is necessary for biofilm accumulation. As a functional consequence, tigecycline induced biofilm formation significantly impaired the up-take of S. epidermidis by mouse macrophage-like cell line J774A.1. Our data provide novel evidence for the molecular basis of antibiotic induced biofilm formation, a phenotype associated with inherently increased antimicrobial tolerance. While this could explain failure of antimicrobial therapies, persistence of S. epidermidis infections in the presence of sub-inhibitory antimicrobials is additionally propelled by biofilm-related impairment of macrophage-mediated pathogen eradication.

  15. HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

    PubMed Central

    Bernard, Mark A.; Han, Xinbing; Inderbitzin, Sonya; Agbim, Ifunanya; Zhao, Hui; Koziel, Henry; Tachado, Souvenir D.

    2014-01-01

    Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients. PMID:25090652

  16. Early growth response-1 protein is induced by JC virus infection and binds and regulates the JC virus promoter

    SciTech Connect

    Romagnoli, Luca; Sariyer, Ilker K.; Tung, Jacqueline; Feliciano, Mariha; Sawaya, Bassel E.; Del Valle, Luis; Ferrante, Pasquale; Khalili, Kamel; Safak, Mahmut; White, Martyn K.

    2008-06-05

    JC virus (JCV) is a human polyomavirus that can emerge from a latent state to cause the cytolytic destruction of oligodendrocytes in the brain resulting in the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Previous studies described a cis-acting transcriptional regulatory element in the JCV non-coding control region (NCCR) that is involved in the response of JCV to cytokines. This consists of a 23 base pair GGA/C rich sequence (GRS) near the replication origin (5112 to + 4) that contains potential binding sites for Sp1 and Egr-1. Gel shift analysis showed that Egr-1, but not Sp1, bound to GRS. Evidence is presented that the GRS gel shift seen on cellular stimulation is due to Egr-1. Thus, TPA-induced GRS gel shift could be blocked by antibody to Egr-1. Further, the TPA-induced GRS DNA/protein complex was isolated and found to contain Egr-1 by Western blot. No other Egr-1 sites were found in the JCV NCCR. Functionally, Egr-1 was found to stimulate transcription of JCV late promoter but not early promoter reporter constructs. Mutation of the Egr-1 site abrogated Egr-1 binding and virus with the mutated Egr-1 site showed markedly reduced VP1 expression and DNA replication. Infection of primary astrocytes by wild-type JCV induced Egr-1 nuclear expression that was maximal at 5-10 days post-infection. Finally, upregulation of Egr-1 was detected in PML by immunohistochemistry. These data suggest that Egr-1 induction may be important in the life cycle of JCV and PML pathogenesis.

  17. Computational identification of conserved transcription factor binding sites upstream of genes induced in rat brain by transient focal ischemic stroke

    PubMed Central

    Pulliam, John V.K.; Xu, Zhenfeng; Ford, Gregory D.; Liu, Cuimei; Li, Yonggang; Stovall, Kyndra; Cannon, Virginetta S.; Tewolde, Teclemichael; Moreno, Carlos S.; Ford, Byron D.

    2013-01-01

    Microarray analysis has been used to understand how gene regulation plays a critical role in neuronal injury, survival and repair following ischemic stroke. To identify the transcriptional regulatory elements responsible for ischemia-induced gene expression, we examined gene expression profiles of rat brains following focal ischemia and performed computational analysis of consensus transcription factor binding sites (TFBS) in the genes of the dataset. In this study, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) stroke and gene transcription in brain tissues following ischemia/reperfusion was examined using Affymetrix GeneChip technology. The CONserved transcription FACtor binding site (CONFAC) software package was used to identify over-represented TFBS in the upstream promoter regions of ischemia-induced genes compared to control datasets. CONFAC identified 12 TFBS that were statistically over-represented from our dataset of ischemia-induced genes, including three members of the Ets-1 family of transcription factors (TFs). Microarray results showed that mRNA for Ets-1 was increased following tMCAO but not pMCAO. Immunohistochemical analysis of Ets-1 protein in rat brains following MCAO showed that Ets-1 was highly expressed in neurons in the brain of sham control animals. Ets-1 protein expression was virtually abolished in injured neurons of the ischemic brain but was unchanged in peri-infarct brain areas. These data indicate that TFs, including Ets-1, may influence neuronal injury following ischemia. These findings could provide important insights into the mechanisms that lead to brain injury and could provide avenues for the development of novel therapies. PMID:23246490

  18. In Vitro Selection of Shape-Changing DNA Nanostructures Capable of Binding-Induced Cargo Release

    PubMed Central

    Oh, Seung Soo; Plakos, Kory; Xiao, Yi; Eisenstein, Michael; Soh, Hyongsok Tom

    2014-01-01

    Many biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate ‘cargo strand’ containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 μM, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications. PMID:24168267

  19. Acidic extracellular pH of tumors induces octamer-binding transcription factor 4 expression in murine fibroblasts in vitro and in vivo

    PubMed Central

    Som, Avik; Bloch, Sharon; Ippolito, Joseph E.; Achilefu, Samuel

    2016-01-01

    Octamer-binding transcription factor 4 (OCT-4) is an important marker of cellular de-differentiation that can be induced by environmental stressors, such as acidity. Here we demonstrate that chronic acidic stress in solid tumors induced OCT-4 expression in fibroblasts and other stromal cells in four tumor models. The results have implications for how tumors utilize pH modulation to recruit associated stromal cells, induce partial reprogramming of tumor-associated stromal cells, and respond to therapy. PMID:27302093

  20. HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

    PubMed Central

    Fiorucci, Gianna; Vaccari, Gabriele; Acconcia, Filippo; Chiarabelli, Cristiano; Leone, Stefano; Noto, Alessia; Horenkamp, Florian A.; Manrique, Santiago; Romeo, Giovanna; Polticelli, Fabio; Geyer, Matthias; Affabris, Elisabetta

    2011-01-01

    Background HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. Methodology/Principal Findings Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. Conclusions Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ. PMID:21886773

  1. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway.

    PubMed

    Kou, Xianjuan; Qi, Shimei; Dai, Wuxing; Luo, Lan; Yin, Zhimin

    2011-08-01

    Arctigenin has been demonstrated to have an anti-inflammatory function, but the precise mechanisms of its action remain to be fully defined. In the present study, we determined the effects of arctigenin on lipopolysaccharide (LPS)-induced production of proinflammatory mediators and the underlying mechanisms involved in RAW264.7 cells. Our results indicated that arctigenin exerted its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity. Arctigenin also significantly reduced the phosphorylation of STAT1 and STAT 3 as well as JAK2 in LPS-stimulated RAW264.7 cells. The inhibitions of STAT1 and STAT 3 by arctigenin prevented their translocation to the nucleus and consequently inhibited expression of iNOS, thereby suppressing the expression of inflammation-associated genes, such as IL-1β, IL-6 and MCP-1, whose promoters contain STAT-binding elements. However, COX-2 expression was slightly inhibited at higher drug concentrations (50 μM). Our data demonstrate that arctigenin inhibits iNOS expression via suppressing JAK-STAT signaling pathway in macrophages.

  2. Cold-inducible RNA-binding protein mediates cold air inducible airway mucin production through TLR4/NF-κB signaling pathway.

    PubMed

    Chen, Lingxiu; Ran, Danhua; Xie, Wenyue; Xu, Qing; Zhou, Xiangdong

    2016-10-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases and cold air stimulation has been shown to be associated with the severity of these diseases. However, the regulatory mechanisms that mediate excessive mucin production under cold stress remain elusive. Recently, the cold-inducible RNA-binding protein (CIRP) has been shown to be markedly induced after exposure to cold air. In this study, we sought to explore the expression of CIRP within bronchial biopsy specimens, the effect on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in cold air stimulation process. We found that CIRP protein expression was significantly increased in patients with COPD and in mice treated with cold air. Moreover, cold air stimulation induced MUC5AC expression in wild-type mice but not in CIRP(-/-) mice. In vitro, cold air stress significantly elevated the transcriptional and protein expression levels of MUC5AC in human bronchial epithelial cells. CIRP, toll-like receptor 4 (TLR4) and phosphorylated NF-κB p65 (p-p65) increased significantly in response to cold stress and CIRP siRNA, TLR4 - neutralizing Ab and a specific inhibitor of NF-κB could attenuated cold stress inducible MUC5AC expression. In addition, CIRP siRNA could hindered the expression levels of TLR4 and p-p65 both induced by cold stress. Taken together, these results suggest that airway epithelial cells constitutively express CIRP in vitro and in vivo. CIRP is responsible for cold-inducible MUC5AC expression by activating TLR4/NF-κB signaling pathway. PMID:27423012

  3. Cold-inducible RNA-binding protein mediates cold air inducible airway mucin production through TLR4/NF-κB signaling pathway.

    PubMed

    Chen, Lingxiu; Ran, Danhua; Xie, Wenyue; Xu, Qing; Zhou, Xiangdong

    2016-10-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases and cold air stimulation has been shown to be associated with the severity of these diseases. However, the regulatory mechanisms that mediate excessive mucin production under cold stress remain elusive. Recently, the cold-inducible RNA-binding protein (CIRP) has been shown to be markedly induced after exposure to cold air. In this study, we sought to explore the expression of CIRP within bronchial biopsy specimens, the effect on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in cold air stimulation process. We found that CIRP protein expression was significantly increased in patients with COPD and in mice treated with cold air. Moreover, cold air stimulation induced MUC5AC expression in wild-type mice but not in CIRP(-/-) mice. In vitro, cold air stress significantly elevated the transcriptional and protein expression levels of MUC5AC in human bronchial epithelial cells. CIRP, toll-like receptor 4 (TLR4) and phosphorylated NF-κB p65 (p-p65) increased significantly in response to cold stress and CIRP siRNA, TLR4 - neutralizing Ab and a specific inhibitor of NF-κB could attenuated cold stress inducible MUC5AC expression. In addition, CIRP siRNA could hindered the expression levels of TLR4 and p-p65 both induced by cold stress. Taken together, these results suggest that airway epithelial cells constitutively express CIRP in vitro and in vivo. CIRP is responsible for cold-inducible MUC5AC expression by activating TLR4/NF-κB signaling pathway.

  4. Binding-induced Stabilization and Assembly of the Phage P22 Tail Accessory Factor gp4

    SciTech Connect

    Olia,A.; Al-Bassam, J.; Winn-Stapley, D.; Joss, L.; Casjens, S.; Cingolani, G.

    2006-01-01

    To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (T{sub m} 34 {sup o}C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1){sub 12}:gp(4){sub 6} assembly intermediate, which is stably populated at 30 {sup o}C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1){sub 12}:gp(4){sub 12} complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.

  5. The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.

    PubMed

    Safaei, Roohangiz; Adams, Preston L; Mathews, Ryan A; Manorek, Gerald; Howell, Stephen B

    2013-08-01

    The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.

  6. The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B

    PubMed Central

    Safaei, Roohangiz; Adams, Preston L.; Mathews, Ryan A.; Manorek, Gerald; Howell, Stephen B.

    2014-01-01

    The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B. PMID:23803742

  7. Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

    PubMed Central

    Diekmann, W; Venis, M A; Robinson, D G

    1995-01-01

    The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific. Images Fig. 1 Fig. 2 Fig. 3 PMID:11607527

  8. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    SciTech Connect

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  9. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of /sup 125/I-fibrinogen to rat platelets

    SciTech Connect

    McGregor, L.; Toor, B.; McGregor, J.L.; Renaud, S.; Clemetson, K.J.

    1984-03-01

    The aggregation to ADP and the binding of /sup 125/I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.

  10. The Molecular Chaperone Binding Protein BiP Prevents Leaf Dehydration-Induced Cellular Homeostasis Disruption

    PubMed Central

    Carvalho, Humberto H.; Brustolini, Otávio J. B.; Pimenta, Maiana R.; Mendes, Giselle C.; Gouveia, Bianca C.; Silva, Priscila A.; Silva, José Cleydson F.; Mota, Clenilso S.; Soares-Ramos, Juliana R. L.; Fontes, Elizabeth P. B.

    2014-01-01

    BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of −1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = −1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = −1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = −1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = −1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = −1.0, −1.7 and −2.0 MPa) and 35S::BiP-4 leaves at ψw = −1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = −1.6 MPa) to respond to drought than that of WT (ψw = −1

  11. The molecular chaperone binding protein BiP prevents leaf dehydration-induced cellular homeostasis disruption.

    PubMed

    Carvalho, Humberto H; Brustolini, Otávio J B; Pimenta, Maiana R; Mendes, Giselle C; Gouveia, Bianca C; Silva, Priscila A; Silva, José Cleydson F; Mota, Clenilso S; Soares-Ramos, Juliana R L; Fontes, Elizabeth P B

    2014-01-01

    BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of -1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = -1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = -1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = -1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = -1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = -1.0, -1.7 and -2.0 MPa) and 35S::BiP-4 leaves at ψw = -1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = -1.6 MPa) to respond to drought than that of WT (ψw = -1.0). Therefore, Bi

  12. Conformational changes in human red cell membrane proteins induced by sugar binding.

    PubMed

    Janoshazi, A; Kifor, G; Solomon, A K

    1991-09-01

    We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions. PMID:1744899

  13. Activating enhancer-binding protein-2α induces cyclooxygenase-2 expression and promotes nasopharyngeal carcinoma growth

    PubMed Central

    Qin, Lijun; Xie, Fangyun; Sun, Rui; Wang, Jingshu; Li, Wenbin; Liu, Tianze; Xiao, Yao; Yu, Wendan; Guo, Wei; Xiong, Yuqing; Qiu, Huijuan; Kang, Tiebang; Huang, Wenlin; Zhao, Chong; Deng, Wuguo

    2015-01-01

    Activating enhancer-binding protein-2α (AP-2α) regulates the expression of many cancer-related genes. Here, we demonstrated a novel mechanism by which AP-2α up-regulated cyclooxygenase-2 (COX-2) expression to promote the growth of nasopharyngeal carcinomas (NPCs). High expression of AP-2α in NPC cell lines and tumor tissues from NPC patients was detected and significantly correlated with COX-2 expression. Overexpression of AP-2α and COX-2 in tumor tissues was associated with advanced tumor stage, clinical progression, and short survival of patients with NPCs. Knockdown of AP-2α by siRNA markedly inhibited COX-2 expression and PGE2 production in NPC cells. Exogenous expression of AP-2α up-regulated the COX-2 and PGE2. Knockdown of AP-2α also significantly suppressed cell proliferation in NPC cells in vitro and tumor growth in a NPC xenograft mouse model. Moreover, we found that p300 played an important role in the AP-2α/COX-2 pathway. AP-2α could co-localize and interact with p300 in NPC cells. Overexpression of the p300, but not its histone acetyltransferase (HAT) domain deletion mutant, promoted the acetylation of AP-2α and its binding on the COX-2 promoter, thereby up-regulated COX-2 expression. Our results indicate that AP-2α activates COX-2 expression to promote NPC growth and suggest that the AP-2α/COX-2 signaling is a potential therapeutic target for NPC treatment. PMID:25669978

  14. Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

    PubMed Central

    Zakaria, Siti Mariam; Frings, Oliver; Fahlén, Sara; Nilsson, Helén; Goodwin, Jacob; von der Lehr, Natalie; Su, Yingtao; Lüscher, Bernhard; Castell, Alina; Larsson, Lars-Gunnar

    2016-01-01

    The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc. PMID:26701207

  15. Inflammatory signals induce the expression of tonicity-responsive enhancer binding protein (TonEBP) in microglia.

    PubMed

    Jeong, Ga Ram; Im, Sun-Kyoung; Bae, Yun-Hee; Park, Eun Su; Jin, Byung Kwan; Kwon, Hyug Moo; Lee, Beom-Joon; Bu, Youngmin; Hur, Eun-Mi; Lee, Byoung Dae

    2016-06-15

    Tonicity-responsive enhancer (TonE) binding protein (TonEBP) is known as an osmosensitive transcription factor that regulates cellular homeostasis during states of hypo- and hypertonic stress. In addition to its role in osmoadaptation, growing lines of evidence suggest that TonEBP might have tonicity-independent functions. In particular, a number of studies suggest that inflammatory stimuli induce the expression and activation of TonEBP in peripheral immune cells. However, whether TonEBP is expressed in microglia, resident immune cells of the central nervous system, is unknown. Here we show that inflammatory signals induce the expression of TonEBP in microglia both in vitro and in vitro. In cultured primary microglia, treatment with lipopolysaccharide (LPS), interferon-γ, and interleukin 4 increased the expression of TonEBP. Moreover, we found that stereotaxic injection of LPS into the substantia nigra region of rat brain increased TonEBP expression in OX-42-positive cells. Furthermore, expression of TonEBP was induced in OX-42-positive cells in a rat model of transient middle cerebral artery occlusion. Together these results show that the expression of TonEBP is regulated by inflammatory signals in mammalian brain, suggesting that TonEBP might play a part during neuroinflammation. PMID:27235345

  16. FAIM-L is an IAP-binding protein that inhibits XIAP ubiquitinylation and protects from Fas-induced apoptosis.

    PubMed

    Moubarak, Rana S; Planells-Ferrer, Laura; Urresti, Jorge; Reix, Stéphanie; Segura, Miguel F; Carriba, Paulina; Marqués-Fernàndez, Fernando; Sole, Carme; Llecha-Cano, Nuria; Lopez-Soriano, Joaquin; Sanchis, Daniel; Yuste, Victor J; Comella, Joan X

    2013-12-01

    The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.

  17. Anti-Inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-κB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit only NF-κB Activation along with Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

    PubMed Central

    Hämäläinen, Mari; Nieminen, Riina; Vuorela, Pia; Heinonen, Marina; Moilanen, Eeva

    2007-01-01

    In inflammation, bacterial products and proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we systematically investigated the effects of 36 naturally occurring flavonoids and related compounds on NO production in macrophages exposed to an inflammatory stimulus (lipopolysaccharide, LPS), and evaluated the mechanisms of action of the effective compounds. Flavone, the isoflavones daidzein and genistein, the flavonols isorhamnetin, kaempferol and quercetin, the flavanone naringenin, and the anthocyanin pelargonidin inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. All eight active compounds inhibited the activation of nuclear factor-κB (NF-κB), which is a significant transcription factor for iNOS. Genistein, kaempferol, quercetin, and daidzein also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1), another important transcription factor for iNOS. The present study characterises the effects and mechanisms of naturally occurring phenolic compounds on iNOS expression and NO production in activated macrophages. The results partially explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds. PMID:18274639

  18. Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

    PubMed

    Sanches, Jéssica de Souza; de Aguiar, Rodrigo Barbosa; Parise, Carolina Bellini; Suzuki, Juliana Mayumi; Chammas, Roger; de Moraes, Jane Zveiter

    2016-04-01

    Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus-dose administration of the antibody. This limitation could be circumvented through the use of anti-idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF-binding immune response generated by an anti-bevacizumab idiotype mAb, 10.D7. The 10.D7 anti-Id mAb vaccination led to detectable levels of VEGF-binding anti-anti-Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7-immunized mice were challenged with B16-F10 tumor cells. Mice immunized with 10.D7 anti-Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7-immunized mice showed increased necrotic areas, decreased CD31-positive vascular density and reduced CD68-positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti-Id therapeutic vaccination maintains stable levels of VEGF-binding antibodies, which might be useful in the control of tumor relapse.

  19. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  20. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  1. The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.

    PubMed Central

    Kvietikova, I; Wenger, R H; Marti, H H; Gassmann, M

    1995-01-01

    The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing. Images PMID:8524640

  2. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

    PubMed Central

    2015-01-01

    Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin. PMID:24635452

  3. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment.

  4. HMGN2 inducibly binds a novel transactivation domain in nuclear PRLr to coordinate Stat5a-mediated transcription.

    PubMed

    Fiorillo, Alyson A; Medler, Terry R; Feeney, Yvonne B; Liu, Yi; Tommerdahl, Kalie L; Clevenger, Charles V

    2011-09-01

    The direct actions of transmembrane receptors within the nucleus remain enigmatic. In this report, we demonstrate that the prolactin receptor (PRLr) localizes to the nucleus where it functions as a coactivator through its interactions with the latent transcription factor signal transducer and activator of transcription 5a (Stat5a) and the high-mobility group N2 protein (HMGN2). We identify a novel transactivation domain within the PRLr that is activated by ligand-induced phosphorylation, an event coupled to HMGN2 binding. The association of the PRLr with HMGN2 enables Stat5a-responsive promoter binding, thus facilitating transcriptional activation and promoting anchorage-independent growth. We propose that HMGN2 serves as a critical regulatory factor in Stat5a-driven gene expression by facilitating the assembly of PRLr/Stat5a onto chromatin and that these events may serve to promote biological events that contribute to a tumorigenic phenotype. Our data imply that phosphorylation may be the molecular switch that activates a cell surface receptor transactivation domain, enabling it to tether chromatin-modifying factors, such as HMGN2, to target promoter regions in a sequence-specific manner.

  5. Surface antigens on cat leukemic cells induced by feline leukemia virus: area density and antibody-binding affinity.

    PubMed

    Boone, C W; Gordin, F; Kawakami, T G

    1973-04-01

    The binding of autologous bovine antibody to feline leukemia virus-induced cell surface antigens (FeCSA) on cat leukemia cells was studied by performing certain titration procedures with a mixture of immune and normal sera labeled with different iodine radioisotopes (paired-label technique). By using plots of titration data which conformed to linear equations derived from the mass action law, we determined the following constants. (i) The density of FeCSA was 2.03 x 10(6) sites per cell, or 5,230 sites per mum(2). (ii) The equilibrium constant of the FeCSA-antibody reaction was 2.67 x 10(7), from which the antibody binding affinity or standard free energy of the FeCSA-antibody bond was determined to be - 10.48 kcal (-43,869.28 J) per mol. The use of the techniques described to measure the concentration of antibody in antiserum, in micrograms per milliliter, is discussed.

  6. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    PubMed

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  7. Keratinocytes-associated chemokines and enzymatically quiescent heparanase induce the binding of resting CD4+ T cells.

    PubMed

    Hershkoviz, R; Marikovsky, M; Gilat, D; Lider, O

    1996-02-01

    Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli. PMID:8601723

  8. HUMAN PAPILLOMAVIRUS E7 ENHANCES HYPOXIA-INDUCIBLE FACTOR 1 MEDIATED TRANSCRIPTION BY INHIBITING BINDING OF HISTONE DEACETYLASES

    PubMed Central

    Bodily, Jason M.; Mehta, Kavi P. M.; Laimins, Laimonis A.

    2010-01-01

    Infection by human papillomaviruses (HPVs) leads to the formation of benign lesions, warts, and in some cases, cervical cancer. The formation of these lesions is dependent upon increased expression of pro-angiogenic factors. Angiogenesis is linked to tissue hypoxia through the activity of the oxygen sensitive hypoxia inducible factor 1α (HIF-1α). Our studies indicate that the HPV E7 protein enhances HIF-1 transcriptional activity while E6 functions to counteract the repressive effects of p53. Both high and low risk HPV E7 proteins were found to bind to HIF-1α through a domain located in the the N terminus. Importantly, the ability of E7 to enhance HIF-1 activity mapped to the C terminus and correlated with the displacement of the histone deacetylases HDAC1, HDAC4, and HDAC7 from HIF-1α by E7. Our findings describe a novel role of the E7 oncoprotein in activating the function of a key transcription factor mediating hypoxic responses by blocking the binding of HDACs. PMID:21148070

  9. Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

    PubMed

    Snopok, Borys; Kruglenko, Ivanna

    2015-05-01

    An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings. PMID:25763411

  10. H2-M polymorphism in mice susceptible to collagen-induced arthritis involves the peptide binding groove

    SciTech Connect

    Walter, W.; Loos, M.; Maeurer, M.J.

    1996-12-31

    The ability to develop type II collagen (CII)-induced arthritis (CIA) in mice is associated with the major histocompatibility I-A gene and with as yet poorly defined regulatory molecules of the major histocompatibility complex (MHC) class II antigen processing and presentation pathway. H2-M molecules are thought to be involved in the loading of antigenic peptides into the MHC class II binding cleft. We sequenced H2-Ma, H2-Mb1, and H2-Mb2 genes from CIA-susceptible and -resistant mouse strains and identified four different Ma and Mb2 alleles, and three different Mb1 alleles defined by polymorphic residues within the predicted peptide binding groove. Most CIA-resistant mouse strains share common Ma, Mb1, and Mb2 alleles. In contrast, H2-M alleles designated Ma-III, Ma-IV, Mb1-III, and Mb2-IV could be exclusively identified in the CIA-susceptible H2{sup r} and H2{sup q} haplotypes, suggesting that allelic H2-M molecules may modulate the composition of different CII peptides loaded onto MHC class II molecules, presumably presenting {open_quotes}arthritogenic{close_quotes} epitopes to T lymphocytes. 42 refs., 4 figs., 3 tabs.

  11. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  12. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  13. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.

  14. Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury

    PubMed Central

    Wang, Guan; Zhang, Jian-ning; Guo, Jia-kui; Cai, Ying; Sun, Hong-sheng; Dong, Kun; Wu, Cheng-gang

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury. PMID:27335561

  15. Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.

    PubMed

    Ohkawara, Bisei; Glinka, Andrei; Niehrs, Christof

    2011-03-15

    The R-Spondin (Rspo) family of secreted Wnt modulators is involved in development and disease and holds therapeutic promise as stem cell growth factors. Despite growing biological importance, their mechanism of action is poorly understood. Here, we show that Rspo3 binds syndecan 4 (Sdc4) and that together they activate Wnt/PCP signaling. In Xenopus embryos, Sdc4 and Rspo3 are essential for two Wnt/PCP-driven processes-gastrulation movements and head cartilage morphogenesis. Rspo3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK. Rspo3 functions by inducing Sdc4-dependent, clathrin-mediated endocytosis. We show that this internalization is essential for PCP signal transduction, suggesting that endocytosis of Wnt-receptor complexes is a key mechanism by which R-spondins promote Wnt signaling.

  16. G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch

    PubMed Central

    Guan, Ai-Jiao; Shen, Meng-Jie; Xiang, Jun-Feng; Zhang, En-Xuan; Li, Qian; Sun, Hong-Xia; Wang, Li-Xia; Xu, Guang-Zhi; Tang, Ya-Lin; Xu, Li-Jin; Gong, Han-Yuan

    2015-01-01

    Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and 1H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition. PMID:25990684

  17. Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

    PubMed

    Sakaguchi, H; Wada, K; Maekawa, M; Watsuji, T; Hagiwara, M

    1999-05-15

    We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning. PMID:10234027

  18. G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch

    NASA Astrophysics Data System (ADS)

    Guan, Ai-Jiao; Shen, Meng-Jie; Xiang, Jun-Feng; Zhang, En-Xuan; Li, Qian; Sun, Hong-Xia; Wang, Li-Xia; Xu, Guang-Zhi; Tang, Ya-Lin; Xu, Li-Jin; Gong, Han-Yuan

    2015-05-01

    Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and 1H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

  19. Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture

    PubMed Central

    Ju, Xiangwu; Yan, Yiwu; Liu, Qiang; Li, Ning; Sheng, Miaomiao; Zhang, Lifang; Li, Xiao; Liang, Zhu; Huang, Fengming; Liu, Kangtai; Zhao, Yan; Zhang, Yanxu; Zou, Zhen; Du, Jianchao; Zhong, Ying; Zhou, Huandi; Yang, Peng; Lu, Huijun; Tian, Mingyao; Li, Dangsheng; Zhang, Jianming

    2015-01-01

    ABSTRACT As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian

  20. Polyhydroxyfullerene binds cadmium ions and alleviates metal-induced oxidative stress in Saccharomyces cerevisiae.

    PubMed

    Pradhan, Arunava; Pinheiro, José Paulo; Seena, Sahadevan; Pascoal, Cláudia; Cássio, Fernanda

    2014-09-01

    The water-soluble polyhydroxyfullerene (PHF) is a functionalized carbon nanomaterial with several industrial and commercial applications. There have been controversial reports on the toxicity and/or antioxidant properties of fullerenes and their derivatives. Conversely, metals have been recognized as toxic mainly due to their ability to induce oxidative stress in living organisms. We investigated the interactive effects of PHF and cadmium ions (Cd) on the model yeast Saccharomyces cerevisiae by exposing cells to Cd (≤5 mg liter(-1)) in the absence or presence of PHF (≤500 mg liter(-1)) at different pHs (5.8 to 6.8). In the absence of Cd, PHF stimulated yeast growth up to 10.4%. Cd inhibited growth up to 79.7%, induced intracellular accumulation of reactive oxygen species (ROS), and promoted plasma membrane disruption in a dose- and pH-dependent manner. The negative effects of Cd on growth were attenuated by the presence of PHF, and maximum growth recovery (53.8%) was obtained at the highest PHF concentration and pH. The coexposure to Cd and PHF decreased ROS accumulation up to 36.7% and membrane disruption up to 30.7% in a dose- and pH-dependent manner. Two mechanisms helped to explain the role of PHF in alleviating Cd toxicity to yeasts: PHF decreased Cd-induced oxidative stress and bound significant amounts of Cd in the extracellular medium, reducing its bioavailability to the cells.

  1. Polyhydroxyfullerene Binds Cadmium Ions and Alleviates Metal-Induced Oxidative Stress in Saccharomyces cerevisiae

    PubMed Central

    Pradhan, Arunava; Pinheiro, José Paulo; Seena, Sahadevan; Pascoal, Cláudia

    2014-01-01

    The water-soluble polyhydroxyfullerene (PHF) is a functionalized carbon nanomaterial with several industrial and commercial applications. There have been controversial reports on the toxicity and/or antioxidant properties of fullerenes and their derivatives. Conversely, metals have been recognized as toxic mainly due to their ability to induce oxidative stress in living organisms. We investigated the interactive effects of PHF and cadmium ions (Cd) on the model yeast Saccharomyces cerevisiae by exposing cells to Cd (≤5 mg liter−1) in the absence or presence of PHF (≤500 mg liter−1) at different pHs (5.8 to 6.8). In the absence of Cd, PHF stimulated yeast growth up to 10.4%. Cd inhibited growth up to 79.7%, induced intracellular accumulation of reactive oxygen species (ROS), and promoted plasma membrane disruption in a dose- and pH-dependent manner. The negative effects of Cd on growth were attenuated by the presence of PHF, and maximum growth recovery (53.8%) was obtained at the highest PHF concentration and pH. The coexposure to Cd and PHF decreased ROS accumulation up to 36.7% and membrane disruption up to 30.7% in a dose- and pH-dependent manner. Two mechanisms helped to explain the role of PHF in alleviating Cd toxicity to yeasts: PHF decreased Cd-induced oxidative stress and bound significant amounts of Cd in the extracellular medium, reducing its bioavailability to the cells. PMID:25038095

  2. Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells

    PubMed Central

    2011-01-01

    Background ATP binding is essential for the bioactivity of several growth factors including nerve growth factor, fibroblast growth factor-2 and brain-derived neurotrophic factor. Vascular endothelial growth factor isoform 165 (VEGF-A165) induces the proliferation of human umbilical vein endothelial cells, however a dependence on ATP-binding is currently unknown. The aim of the present study was to determine if ATP binding is essential for the bioactivity of VEGF-A165. Results We found evidence that ATP binding toVEGF-A165 induced a conformational change in the secondary structure of the growth factor. This binding appears to be significant at the biological level, as we found evidence that nanomolar levels of ATP (4-8 nm) are required for the VEGF-A165-induced proliferation of human umbilical vein endothelial cells. At these levels, purinergic signaling by ATP via P2 receptors can be excluded. Addition of alkaline phosphate to cell culture lowered the ATP concentration in the cell culture medium to 1.8 nM and inhibited cell proliferation. Conclusions We propose that proliferation of endothelial cells is induced by a VEGF-A165-ATP complex, rather than VEGF-A165 alone. PMID:21619628

  3. Binding induced colocalization activated hybridization chain reaction on the surface of magnetic nanobead for sensitive detection of adenosine.

    PubMed

    Feng, Chunjing; Hou, Zhun; Jiang, Wei; Sang, Lihong; Wang, Lei

    2016-12-15

    Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10(-7)mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.

  4. Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal

    PubMed Central

    Lee, Minsup; Li, Songhua; Sato, Kota; Jin, Minghao

    2016-01-01

    Purpose Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp−/− retina. Methods Wild-type and Irbp−/− mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer. Results Content of atRAL in Irbp−/− retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP. Conclusions Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the

  5. Hydroxychloroquine protects melanocytes from autoantibody-induced injury by reducing the binding of antigen-antibody complexes.

    PubMed

    Li, Dong-Guang; Hu, Wen-Zhi; Ma, Hui-Jun; Liu, Wen; Yang, Qing-Qi; Zhao, Guang

    2016-08-01

    Vitiligo is a polygenic autoimmune disorder characterized by loss of pigmentation due to melanocyte destruction. Hydroxychloroquine (HCQ) is an effective immunosuppressant widely used in the treatment of autoimmune disorders. As generalized vitiligo (GV) is commonly considered to be a T cell and autoantibody-induced immune disorder, the present study aimed to determine whether HCQ protects melanocytes from autoantibody‑induced disruption. Anti‑melanocyte antibodies were obtained from the serum of patients with progressive GV and the effects of HCQ on prevent the autoantibody‑induced disruption of melanocytes was observed. Cell‑based ELISA, indirect immunofluorescence and western blotting were used to analyze the autoantibody content of sera samples obtained from 32 patients with progressive GV. The cytotoxicity of HCQ was detected by MTT assay, and 1 µg/ml HCQ was applied to human primary melanocytes (HMCs) to examine whether it could exert protective effects against autoantibody‑induced immune injury. Flow cytometry was used to measure autoantibody binding to the surface of HMCs. Complement‑dependent cytotoxicity (CDC) and antibody‑dependent cell‑mediated cytotoxicity (ADCC) were monitored by MTT and lactate dehydrogenase‑releasing assays. The concentration of autoantibodies in sera samples taken from GV patients was significantly higher than in controls, particularly in patients who had >10% of their body surface affected by vitiligo. The majority of the autoantibodies presented in the HMCs and human keratinocytes (HKCs) and were predominantly localized to the cell surface and cytoplasm. The molecular weights of the autoantigens were identified as 30, 37‑39, 42, 53, 60‑75, 90, 100, 110, and 126 kDa; the 30 kDa protein was observed only in HMCs. The addition of HCQ at a concentration of 1 µg/ml produced no significant cytotoxicity in HMCs and was demonstrated to reduce the binding of GV immunoglobulin G (IgG) to the surface of HMCs

  6. Inhibitor of DNA Binding 1 Is Induced during Kidney Ischemia-Reperfusion and Is Critical for the Induction of Hypoxia-Inducible Factor-1α

    PubMed Central

    Wen, Dan; Zou, Yan-Fang; Gao, Yao-Hui; Zhao, Qian; Xie, Yin-Yin; Shen, Ping-Yan; Xu, Yao-Wen; Xu, Jing; Chen, Yong-Xi; Feng, Xiao-Bei; Shi, Hao; Zhang, Wen

    2016-01-01

    In this study, rat models of acute kidney injury (AKI) induced by renal ischemia-reperfusion (I/R) and HK-2 cell models of hypoxia-reoxygenation (H/R) were established to investigate the expression of inhibitor of DNA binding 1 (ID1) in AKI, and the regulation relationship between ID1 and hypoxia-inducible factor 1 alpha (HIF-1α). Through western blot, quantitative real-time PCR, immunohistochemistry, and other experiment methods, the induction of ID1 after renal I/R in vivo was observed, which was expressed mainly in renal tubular epithelial cells (TECs). ID1 expression was upregulated in in vitro H/R models at both the protein and mRNA levels. Via RNAi, it was found that ID1 induction was inhibited with silencing of HIF-1α. Moreover, the suppression of ID1 mRNA expression could lead to decreased expression and transcription of HIF-1α during hypoxia and reoxygenation. In addition, it was demonstrated that both ID1 and HIF-1α can regulate the transcription of twist. This study demonstrated that ID1 is induced in renal TECs during I/R and can regulate the transcription and expression of HIF-1α. PMID:27127787

  7. Gedunin Binds to Myeloid Differentiation Protein 2 and Impairs Lipopolysaccharide-Induced Toll-Like Receptor 4 Signaling in Macrophages.

    PubMed

    Borges, Perla Villani; Moret, Katelim Hottz; Maya-Monteiro, Clarissa Menezes; Souza-Silva, Franklin; Alves, Carlos Roberto; Batista, Paulo Ricardo; Caffarena, Ernesto Raúl; Pacheco, Patrícia; Henriques, Maria das Graças; Penido, Carmen

    2015-11-01

    Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the cluster of differentiation 14/Toll-like receptor 4/myeloid differentiation protein 2 (MD-2) complex. In this study, we investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as a heat shock protein Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild-type (WT) macrophages with gedunin (0.01-100 µM, noncytotoxic concentrations) inhibited LPS (50 ng/ml)-induced calcium influx, tumor necrosis factor-α, and nitric oxide production in a concentration-dependent manner. The selective effect of gedunin on MyD88-adapter-like/myeloid differentiation primary response 88- and TRIF-related adaptor molecule/TIR domain-containing adapter-inducing interferon-β-dependent signaling pathways was further investigated. The pretreatment of WT, TIR domain-containing adapter-inducing interferon-β knockout, and MyD88 adapter-like knockout macrophages with gedunin (10 µM) significantly inhibited LPS (50 ng/ml)-induced tumor necrosis factor-α and interleukin-6 production, at 6 hours and 24 hours, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 µM) inhibited LPS-induced prostaglandin E2 production, cyclooxygenase-2 expression, and nuclear factor κB translocation into the nucleus of WT macrophages, demonstrating a wide-range effect of this chemical compound. In addition to the ability to inhibit LPS-induced proinflammatory mediators, gedunin also triggered anti-inflammatory factors interleukin-10, heme oxygenase-1, and Hsp70 in macrophages stimulated or not with LPS. In silico modeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further confirmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking

  8. Studies of binding of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to fibroblast growth factor inducible 14 (Fn14).

    PubMed

    Fick, Andrea; Lang, Isabell; Schäfer, Viktoria; Seher, Axel; Trebing, Johannes; Weisenberger, Daniela; Wajant, Harald

    2012-01-01

    To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ∼100-fold higher than Fn14. Thus, although ∼25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response. PMID:22081603

  9. Calreticulin Binds to Fas Ligand and Inhibits Neuronal Cell Apoptosis Induced by Ischemia-Reperfusion Injury

    PubMed Central

    Chen, Beilei; Wu, Zhengzheng; Xu, Jun; Xu, Yun

    2015-01-01

    Background. Calreticulin (CRT) can bind to Fas ligand (FasL) and inhibit Fas/FasL-mediated apoptosis of Jurkat T cells. However, its effect on neuronal cell apoptosis has not been investigated. Purpose. We aimed to evaluate the neuroprotective effect of CRT following ischemia-reperfusion injury (IRI). Methods. Mice underwent middle cerebral artery occlusion (MCAO) and SH-SY5Y cells subjected to oxygen glucose deprivation (OGD) were used as models for IRI. The CRT protein level was detected by Western blotting, and mRNA expression of CRT, caspase-3, and caspase-8 was measured by real-time PCR. Immunofluorescence was used to assess the localization of CRT and FasL. The interaction of CRT with FasL was verified by coimmunoprecipitation. SH-SY5Y cell viability was determined by MTT assay, and cell apoptosis was assessed by flow cytometry. The measurement of caspase-8 and caspase-3 activity was carried out using caspase activity assay kits. Results. After IRI, CRT was upregulated on the neuron surface and bound to FasL, leading to increased viability of OGD-exposed SH-SY5Y cells and decreased activity of caspase-8 and caspase-3. Conclusions. This study for the first time revealed that increased CRT inhibited Fas/FasL-mediated neuronal cell apoptosis during the early stage of ischemic stroke, suggesting it to be a potential protector activated soon after IRI. PMID:26583143

  10. Rational Optimization of Conformational Effects Induced By Hydrocarbon Staples in Peptides and their Binding Interfaces

    NASA Astrophysics Data System (ADS)

    Lama, Dilraj; Quah, Soo T.; Verma, Chandra S.; Lakshminarayanan, Rajamani; Beuerman, Roger W.; Lane, David P.; Brown, Christopher J.

    2013-12-01

    eIF4E is frequently over-expressed in different cancers and causes increased translation of oncogenic proteins via deregulated cap-dependent translation. Inhibitors of the eIF4E:eIF4G interactions represent an approach that would normalize cap-dependent translation. Stapled peptides represent an emerging class of therapeutics that can target protein: protein interactions. We present here molecular dynamics simulations for a set of rationally designed stapled peptides in solution and in complex with eIF4E, supported with biophysical and crystallographic data. Clustering of the simulated structures revealed the favoured conformational states of the stapled peptides in their bound or free forms in solution. Identifying these populations has allowed us to design peptides with improved affinities by introducing mutations into the peptide sequence to alter their conformational distributions. These studies emphasise the effects that engineered mutations have on the conformations of free and bound peptides, and illustrate that both states must be considered in efforts to attain high affinity binding.

  11. Myrosinase-binding proteins are derived from a large wound-inducible and repetitive transcript.

    PubMed

    Taipalensuu, J; Falk, A; Ek, B; Rask, L

    1997-02-01

    Several non-myrosinase proteins have been found in association with some of the myrosinases extracted from rape (Brassica napus) seed. Most of these proteins seemed to belong to a large family of proteins ranging in size over approximately 30-110 kDa, namely the myrosinase-binding protein (MBP) family. Potentially all of these MBPs might be derived from a single large precursor, encoded by a 3.3-kb transcript. This transcript coded for a 99-kDa glycine-rich protein with a highly repetitive structure. The mature 50-kDa and 52-kDa MBP amino-terminal was located 255 amino acids from the putative initiation methionine. Also, a more divergently related transcript, the protein product of which was unknown, has been cloned. However, the largest open reading frame suggested a proline-rich protein. While this transcript seemed to be expressed predominantly in seeds, the MBP transcripts were expressed in several tissues and also exhibited a responsiveness to wounding and methyl jasmonate. Both proteins exhibited significant similarities to lectins from Artocarpus integer and from Maclura pomifera.

  12. Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase.

    PubMed

    Theissen, Bettina; Karow, Anne R; Köhler, Jürgen; Gubaev, Airat; Klostermeier, Dagmar

    2008-01-15

    RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.

  13. Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury.

    PubMed

    De León, M; Welcher, A A; Nahin, R H; Liu, Y; Ruda, M A; Shooter, E M; Molina, C A

    1996-05-01

    Peripheral nerve trauma induces the expression of genes presumed to be involved in the process of nerve degeneration and repair. In the present study, an in vivo paradigm was employed to identify molecules which may have important roles in these processes. A cDNA library was constructed with RNA extracted from rat dorsal root ganglia (DRG) 3 days after a sciatic nerve crush. After differential hybridization to this library, several cDNAs were identified that encoded mRNAs that were upregulated in the DRG ipsilateral to the crush injury, as opposed to the contralateral or naive DRG. Approximately 0.15% of all the clones screened were found to be induced. This report presents the types of induced sequences identified and characterizes one of them, DA11. The 0.7 kb DA11 full length cDNA clone contains a 405 nucleotide open reading frame that encodes a putative protein of 15.2 kDa (135 amino acid residues) and is a member of the family of fatty acid binding proteins (FABP). The DA11 protein differs by one amino acid residue from the sequence of the C-FAPB protein and by eight residues from the sequence of mal1, proteins found in rat and mouse skin, respectively. Northern and Western blot analyses showed that the DA11 mRNA and protein were induced in the injured DRG. Furthermore, studies using antibodies generated against DA11 found that the DA11-like immunoreactivity was more pronounced in the nuclei of neurons located in the DRG ipsilateral to the sciatic cut than those located in the contralateral DRG. The induction of DA11 mRNA and protein in DRG neurons suggests, for the first time, the involvement of a neuronal FABP in the process of degeneration and repair in the nervous system.

  14. Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein.

    PubMed

    Ravon, D M; Citarella, F; Lubbers, Y T; Pascucci, B; Hack, C E

    1995-12-01

    In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the

  15. Arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair

    SciTech Connect

    Sun, Xi; Zhou, Xixi; Du, Libo; Liu, Wenlan; Liu, Yang; Hudson, Laurie G.; Liu, Ke Jian

    2014-01-15

    Inhibition of DNA repair is a recognized mechanism for arsenic enhancement of ultraviolet radiation-induced DNA damage and carcinogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger DNA repair protein, has been identified as a sensitive molecular target for arsenic. The zinc finger domains of PARP-1 protein function as a critical structure in DNA recognition and binding. Since cellular poly(ADP-ribosyl)ation capacity has been positively correlated with zinc status in cells, we hypothesize that arsenite binding-induced zinc loss from PARP-1 is equivalent to zinc deficiency in reducing PARP-1 activity, leading to inhibition of DNA repair. To test this hypothesis, we compared the effects of arsenite exposure with zinc deficiency, created by using the membrane-permeable zinc chelator TPEN, on 8-OHdG formation, PARP-1 activity and zinc binding to PARP-1 in HaCat cells. Our results show that arsenite exposure and zinc deficiency had similar effects on PARP-1 protein, whereas supplemental zinc reversed these effects. To investigate the molecular mechanism of zinc loss induced by arsenite, ICP-AES, near UV spectroscopy, fluorescence, and circular dichroism spectroscopy were utilized to examine arsenite binding and occupation of a peptide representing the first zinc finger of PARP-1. We found that arsenite binding as well as zinc loss altered the conformation of zinc finger structure which functionally leads to PARP-1 inhibition. These findings suggest that arsenite binding to PARP-1 protein created similar adverse biological effects as zinc deficiency, which establishes the molecular mechanism for zinc supplementation as a potentially effective treatment to reverse the detrimental outcomes of arsenic exposure. - Highlights: • Arsenite binding is equivalent to zinc deficiency in reducing PARP-1 function. • Zinc reverses arsenic inhibition of PARP-1 activity and enhancement of DNA damage. • Arsenite binding and zinc loss alter the conformation of zinc finger

  16. Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

    PubMed

    Pesiri, Valeria; La Rosa, Piergiorgio; Stano, Pasquale; Acconcia, Filippo

    2013-06-15

    Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

  17. Zerumbone, a ginger sesquiterpene, induces apoptosis and autophagy in human hormone-refractory prostate cancers through tubulin binding and crosstalk between endoplasmic reticulum stress and mitochondrial insult.

    PubMed

    Chan, Mei-Ling; Liang, Jui-Wei; Hsu, Lih-Ching; Chang, Wei-Ling; Lee, Shoei-Sheng; Guh, Jih-Hwa

    2015-11-01

    Zerumbone, a natural monocyclic sesquiterpene, is the main component of the tropical plant Zingiber zerumbet Smith. Zerumbone induced antiproliferative and apoptotic effects against PC-3 and DU-145, two human hormone-refractory prostate cancer (HRPC) cell lines. Zerumbone inhibited microtubule assembly and induced an increase of MPM-2 expression (specific recognition of mitotic proteins). It also caused an increase of phosphorylation of Bcl-2 and Bcl-xL, two key events in tubulin-binding effect, indicating tubulin-binding capability and mitotic arrest to zerumbone action. Furthermore, zerumbone induced several cellular effects distinct from tubulin-binding properties. First, zerumbone significantly increased, while paclitaxel (as a tubulin-binding control) decreased, Mcl-1 protein expression. Second, paclitaxel but not zerumbone induced Cdk1 activity. Third, zerumbone other than paclitaxel induced Cdc25C downregulation. The data suggest that, in addition to targeting tubulin/microtubule, zerumbone may act on other targets for signaling transduction. Zerumbone induced mitochondrial damage and endoplasmic reticulum (ER) stress as evidenced by the loss of mitochondrial membrane potential and upregulation of GRP-78 and CHOP/GADD153 expression. Zerumbone induced an increase of intracellular Ca(2+) levels, a crosstalk marker between ER stress and mitochondrial insult, associated with the formation of active calpain I fragment. It induced apoptosis through a caspase-dependent way and caused autophagy as evidenced by dramatic LC3-II formation. In summary, the data suggest that zerumbone is a multiple targeting compound that inhibits tubulin assembly and induces a crosstalk between ER stress and mitochondrial insult, leading to apoptosis and autophagy in HRPCs.

  18. Zerumbone, a ginger sesquiterpene, induces apoptosis and autophagy in human hormone-refractory prostate cancers through tubulin binding and crosstalk between endoplasmic reticulum stress and mitochondrial insult.

    PubMed

    Chan, Mei-Ling; Liang, Jui-Wei; Hsu, Lih-Ching; Chang, Wei-Ling; Lee, Shoei-Sheng; Guh, Jih-Hwa

    2015-11-01

    Zerumbone, a natural monocyclic sesquiterpene, is the main component of the tropical plant Zingiber zerumbet Smith. Zerumbone induced antiproliferative and apoptotic effects against PC-3 and DU-145, two human hormone-refractory prostate cancer (HRPC) cell lines. Zerumbone inhibited microtubule assembly and induced an increase of MPM-2 expression (specific recognition of mitotic proteins). It also caused an increase of phosphorylation of Bcl-2 and Bcl-xL, two key events in tubulin-binding effect, indicating tubulin-binding capability and mitotic arrest to zerumbone action. Furthermore, zerumbone induced several cellular effects distinct from tubulin-binding properties. First, zerumbone significantly increased, while paclitaxel (as a tubulin-binding control) decreased, Mcl-1 protein expression. Second, paclitaxel but not zerumbone induced Cdk1 activity. Third, zerumbone other than paclitaxel induced Cdc25C downregulation. The data suggest that, in addition to targeting tubulin/microtubule, zerumbone may act on other targets for signaling transduction. Zerumbone induced mitochondrial damage and endoplasmic reticulum (ER) stress as evidenced by the loss of mitochondrial membrane potential and upregulation of GRP-78 and CHOP/GADD153 expression. Zerumbone induced an increase of intracellular Ca(2+) levels, a crosstalk marker between ER stress and mitochondrial insult, associated with the formation of active calpain I fragment. It induced apoptosis through a caspase-dependent way and caused autophagy as evidenced by dramatic LC3-II formation. In summary, the data suggest that zerumbone is a multiple targeting compound that inhibits tubulin assembly and induces a crosstalk between ER stress and mitochondrial insult, leading to apoptosis and autophagy in HRPCs. PMID:26246051

  19. Undecylprodigiosin Induced Apoptosis in P388 Cancer Cells Is Associated with Its Binding to Ribosome

    PubMed Central

    Liu, Ping; Wang, Yuan-yuan; Qi, Xin; Gu, QianQun; Geng, Meiyu; Li, Jing

    2013-01-01

    Prodigiosins (PGs) are a family of natural red pigments with anticancer activity, and one member of the family has entered clinical phase II trials. However, the anticancer mechanisms of PGs remain largely unclear. This study was designed to investigate the molecular basis of anticancer activity of UP, a derivative of PGs, in P388 cells. By introducing pharmacological inhibitors and utilizing a variety of analytical approaches including western blotting, flow cytometry and confocal laser microscopy, we found that UP inhibited proliferation of P388 via arresting cells at G2/M phase and inducing cells apoptosis, which was related to the activation of P38, JNK rather than ERK1/2 signaling. ROS regeneration and acidification in cells appear not involved in UP induced apoptosis. Furthermore, utilizing mass spectrometry, sucrose density gradient fractionation and immunofluorescence staining, we discovered that UP was apparently located at ribosome. These results together indicate that ribosome may be the potential target of UP in cancer cells, which opened a new avenue in delineating the anticancer mechanism of PGs. PMID:23799011

  20. Effects of corticosterone pellets on baseline and stress-induced corticosterone and corticosteroid-binding-globulin.

    PubMed

    Müller, Claudia; Almasi, Bettina; Roulin, Alexandre; Breuner, Creagh W; Jenni-Eiermann, Susanne; Jenni, Lukas

    2009-01-01

    Exogenous administration of glucocorticoids is a widely used and efficient tool to investigate the effects of elevated concentrations of these hormones in field studies. Because the effects of corticosterone are dose and duration-dependent, the exact course of plasma corticosterone levels after exogenous administration needs to be known. We tested the performance of self-degradable corticosterone pellets (implanted under the skin) in elevating plasma corticosterone levels. We monitored baseline (sampled within 3min after capture) total corticosterone levels and investigated potential interactions with corticosteroid-binding-globulin (CBG) capacity and the endogenous corticosterone response to handling in Eurasian kestrel Falco tinnunculus and barn owl Tyto alba nestlings. Corticosterone pellets designed for a 7-day-release in rodents elevated circulating baseline total corticosterone during only 2-3 days compared to placebo-nestlings. Highest levels occurred 1-2days after implantation and levels decreased strongly thereafter. CBG capacity was also increased, resulting in a smaller, but still significant, increase in baseline free corticosterone levels. The release of endogenous corticosterone as a response to handling was strong in placebo-nestlings, but absent 2 and 8 days after corticosterone pellet implantation. This indicates a potential shut-down of the hypothalamo-pituitary-adrenal axis after the 2-3 days of elevated baseline corticosterone levels. 20 days after pellet implantation, the endogenous corticosterone response to handling of nestlings implanted with corticosterone pellets attained similar levels as in placebo-nestlings. Self-degradable pellets proved to be an efficient tool to artificially elevate circulating baseline corticosterone especially in field studies, requiring only one intervention. The resulting peak-like elevation of circulating corticosterone, the concomitant elevation of CBG capacity, and the absence of an endogenous corticosterone

  1. Effects of corticosterone pellets on baseline and stress-induced corticosterone and corticosteroid-binding-globulin.

    PubMed

    Müller, Claudia; Almasi, Bettina; Roulin, Alexandre; Breuner, Creagh W; Jenni-Eiermann, Susanne; Jenni, Lukas

    2009-01-01

    Exogenous administration of glucocorticoids is a widely used and efficient tool to investigate the effects of elevated concentrations of these hormones in field studies. Because the effects of corticosterone are dose and duration-dependent, the exact course of plasma corticosterone levels after exogenous administration needs to be known. We tested the performance of self-degradable corticosterone pellets (implanted under the skin) in elevating plasma corticosterone levels. We monitored baseline (sampled within 3min after capture) total corticosterone levels and investigated potential interactions with corticosteroid-binding-globulin (CBG) capacity and the endogenous corticosterone response to handling in Eurasian kestrel Falco tinnunculus and barn owl Tyto alba nestlings. Corticosterone pellets designed for a 7-day-release in rodents elevated circulating baseline total corticosterone during only 2-3 days compared to placebo-nestlings. Highest levels occurred 1-2days after implantation and levels decreased strongly thereafter. CBG capacity was also increased, resulting in a smaller, but still significant, increase in baseline free corticosterone levels. The release of endogenous corticosterone as a response to handling was strong in placebo-nestlings, but absent 2 and 8 days after corticosterone pellet implantation. This indicates a potential shut-down of the hypothalamo-pituitary-adrenal axis after the 2-3 days of elevated baseline corticosterone levels. 20 days after pellet implantation, the endogenous corticosterone response to handling of nestlings implanted with corticosterone pellets attained similar levels as in placebo-nestlings. Self-degradable pellets proved to be an efficient tool to artificially elevate circulating baseline corticosterone especially in field studies, requiring only one intervention. The resulting peak-like elevation of circulating corticosterone, the concomitant elevation of CBG capacity, and the absence of an endogenous corticosterone

  2. Purification and molecular cloning of an inducible gram-negative bacteria-