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Sample records for inhibits beta-catenin binding

  1. Inhibition of {beta}-catenin-mediated transactivation by flavanone in AGS gastric cancer cells

    SciTech Connect

    Park, Chi Hoon; Hahm, Eun Ryeong; Lee, Ju Hyung; Jung, Kyung Chae; Yang, Chul Hak . E-mail: chulyang@plaza.snu.ac.kr

    2005-06-17

    Recently, data which prove that Wnt pathway activation may be an early event in multistep carcinogenesis in the stomach have been accumulating. We examined the effect of flavanone against {beta}-catenin/Tcf signaling in AGS gastric cancer cells. Reporter gene assay showed that flavanone inhibited {beta}-catenin/Tcf signaling efficiently. In addition, the inhibition of {beta}-catenin/Tcf signaling by flavanone in HEK293 cells transiently transfected with constitutively mutant {beta}-catenin gene, whose product is not phosphorylated by GSK3{beta}, indicates that its inhibitory mechanism was related to {beta}-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that there is no change of {beta}-catenin distribution and of nuclear {beta}-catenin levels through flavanone. In addition, the binding of Tcf complexes to DNA is not influenced by flavanone. The {beta}-catenin/Tcf transcriptional target gene cyclinD1 was downregulated by flavanone. These data suggest that flavanone inhibits the transcription of {beta}-catenin/Tcf responsive genes, by modulating Tcf activity without disrupting {beta}-catenin/Tcf complex formation.

  2. Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/{beta}-catenin signaling pathway in HCT116 cells

    SciTech Connect

    Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju; Kim, Chul Young; Nho, Chu Won

    2012-11-16

    Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

  3. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.

    PubMed

    Chitalia, Vipul C; Foy, Rebecca L; Bachschmid, Markus M; Zeng, Liling; Panchenko, Maria V; Zhou, Mina I; Bharti, Ajit; Seldin, David C; Lecker, Stewart H; Dominguez, Isabel; Cohen, Herbert T

    2008-10-01

    The von Hippel-Lindau protein pVHL suppresses renal tumorigenesis in part by promoting the degradation of hypoxia-inducible HIF-alpha transcription factors; additional mechanisms have been proposed. pVHL also stabilizes the plant homeodomain protein Jade-1, which is a candidate renal tumour suppressor that may correlate with renal cancer risk. Here we show that Jade-1 binds the oncoprotein beta-catenin in Wnt-responsive fashion. Moreover, Jade-1 destabilizes wild-type beta-catenin but not a cancer-causing form of beta-catenin. Whereas the well-established beta-catenin E3 ubiquitin ligase component beta-TrCP ubiquitylates only phosphorylated beta-catenin, Jade-1 ubiquitylates both phosphorylated and non-phosphorylated beta-catenin and therefore regulates canonical Wnt signalling in both Wnt-off and Wnt-on phases. Thus, the different characteristics of beta-TrCP and Jade-1 may ensure optimal Wnt pathway regulation. Furthermore, pVHL downregulates beta-catenin in a Jade-1-dependent manner and inhibits Wnt signalling, supporting a role for Jade-1 and Wnt signalling in renal tumorigenesis. The pVHL tumour suppressor and the Wnt tumorigenesis pathway are therefore directly linked through Jade-1.

  4. MiR-214 inhibits cell growth in hepatocellular carcinoma through suppression of {beta}-catenin

    SciTech Connect

    Wang, Xiaojun; Chen, Ji; Li, Feng; Lin, Yanting; Zhang, Xiaoping; Lv, Zhongwei; Jiang, Jiaji

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer miR-214 is frequently downregulated in human HCC cell lines and tissues. Black-Right-Pointing-Pointer miR-214 overexpression inhibits HCC cell growth in vitro and in vivo. Black-Right-Pointing-Pointer miR-214 directly targets {beta}-catenin 3 Prime -UTR in HCC cells. Black-Right-Pointing-Pointer miR-214 regulates {beta}-catenin downstream signaling molecules. -- Abstract: Mounting evidence has shown that microRNAs (miRNAs) are implicated in carcinogenesis and can function as oncogenes or tumor suppressor genes in human cancers. Recent profile studies of miRNA expression have documented a deregulation of miRNA (miR-214) in hepatocellular carcinoma (HCC). However, its potential functions and underlying mechanisms in hepatocarcinogenesis remain largely unknown. Here, we confirmed that miR-214 is significantly downregulated in HCC cells and specimens. Ectopic overexpression of miR-214 inhibited proliferation of HCC cells in vitro and tumorigenicity in vivo. Further studies revealed that miR-214 could directly target the 3 Prime -untranslated region (3 Prime -UTR) of {beta}-catenin mRNA and suppress its protein expression. Similar to the restoring miR-214 expression, {beta}-catenin downregulation inhibited cell growth, whereas restoring the {beta}-catenin expression abolished the function of miR-214. Moreover, miR-214-mediated reduction of {beta}-catenin resulted in suppression of several downstream genes including c-Myc, cyclinD1, TCF-1, and LEF-1. These findings indicate that miR-214 serves as tumor suppressor and plays substantial roles in inhibiting the tumorigenesis of HCC through suppression of {beta}-catenin. Given these, miR-214 may serve as a useful prognostic or therapeutic target for treatment of HCC.

  5. {beta}-Catenin mediates the anti-adipogenic effect of baicalin

    SciTech Connect

    Lee, Haeyong; Bae, Sungmin; Kim, Kijeong; Kim, Wonyong; Chung, Sang-In; Yoon, Yoosik

    2010-08-06

    Research highlights: {yields} Baicalin maintains the levels of {beta}-Catenin during adipogenesis. {yields} {beta}-Catenin mediates the anti-adipogenic effect of baicalin. {yields} Baicalin maintains the WNT/{beta}-Catenin pathway during adipogenesis. -- Abstract: {beta}-Catenin reportedly inhibits adipogenesis through the down-regulations of peroxisome proliferator-activated receptor (PPAR){gamma} and CCAAT/enhancer binding protein (C/EBP){alpha}. We report that baicalin, a natural flavonoid compound, inhibits adipogenesis by modulating {beta}-Catenin. During 3T3-L1 cell adipogenesis, {beta}-Catenin was down-regulated, but baicalin treatment maintained {beta}-Catenin expression. Anti-adipogenic effects of baicalin were significantly attenuated by {beta}-Catenin siRNA transfection. {beta}-Catenin siRNA rescued the reduced expressions of PPAR{gamma}, C/EBP{alpha}, fatty acid binding protein 4 and lipoprotein lipase by baicalin. Furthermore, baicalin modulated members of the WNT/{beta}-Catenin pathway by maintaining the expressions of low-density lipoprotein receptor-related protein 6, disheveled (DVL)2 and DVL3. These findings suggest that {beta}-Catenin mediates the anti-adipogenic effects of baicalin.

  6. The nonsteroidal anti-inflammatory drug, nabumetone, differentially inhibits beta-catenin signaling in the MIN mouse and azoxymethane-treated rat models of colon carcinogenesis.

    PubMed

    Roy, Hemant K; Karolski, William J; Wali, Ramesh K; Ratashak, Anne; Hart, John; Smyrk, Thomas C

    2005-01-20

    The mechanisms through which beta-catenin signaling is inhibited during colorectal cancer chemoprevention by nonsteroidal anti-inflammatory agents is incompletely understood. We report that nabumetone decreased uninvolved intestinal mucosal beta-catenin levels in the MIN mouse with a concomitant increase in glycogen synthase kinase (GSK)-3beta levels, an enzyme that targets beta-catenin for destruction. However, in the azoxymethane-treated rat, where beta-catenin is frequently rendered GSK-3beta-insensitive, nabumetone failed to alter beta-catenin levels but did decrease beta-catenin nuclear localization and transcriptional activity as gauged by cyclin D1. In conclusion, we demonstrate that the differential mechanisms for beta-catenin suppression may be determined, at least partly, by GSK-3beta.

  7. Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis

    PubMed Central

    Leal, Letícia F.; Bueno, Ana Carolina; Gomes, Débora C.; Abduch, Rafael; de Castro, Margaret; Antonini, Sonir R.

    2015-01-01

    Background To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. Aim To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Methods Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5–200 μM) for 24–96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). Results In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Conclusions Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC. PMID:26515592

  8. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

    SciTech Connect

    Cho, Il-Rae; Koh, Sang Seok; Malilas, Waraporn; Srisuttee, Ratakorn; Moon, Jeong; Choi, Young-Whan; Horio, Yoshiyuki; Oh, Sangtaek; Chung, Young-Hwa

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1

  9. beta-Catenin/TCF pathway plays a vital role in selenium induced-growth inhibition and apoptosis in esophageal squamous cell carcinoma (ESCC) cells.

    PubMed

    Zhang, Wei; Yan, Shuang; Liu, Mei; Zhang, Guo; Yang, Shangbin; He, Shun; Bai, Jinfeng; Quan, Lanping; Zhu, Hongxia; Dong, Yan; Xu, Ningzhi

    2010-10-01

    Epidemiological and experimental studies have indicated selenium could reduce the risk of some cancers. In our present study, growth inhibition and apoptosis were detected upon methylseleninic acid (MSA) treatment in human esophageal squamous cell carcinoma cell lines EC9706 and KYSE150. MSA reduced beta-catenin protein levels, while there was no significant change observed on transcriptional levels. Moreover, we found MSA accelerated the degradation of beta-catenin and activated glycogen synthase kinase 3beta (GSK-3beta). Some targets of beta-catenin/TCF pathway and apoptosis-related genes altered after MSA treatment. Notably, utilizing the inducible 293-TR/beta-catenin cell line, we found the apoptotic phenotypes induced by MSA were partially reversed by the overexpression of beta-catenin. Overall, our data indicate the effects induced by MSA in ESCC cells may act on the inhibition of beta-catenin/TCF pathway.

  10. Negative regulation of {beta}-catenin/Tcf signaling by naringenin in AGS gastric cancer cell

    SciTech Connect

    Lee, Ju Hyung; Park, Chi Hoon; Jung, Kyung Chae; Rhee, Ho Sung; Yang, Chul Hak . E-mail: chulyang@plaza.snu.ac.kr

    2005-09-30

    Functional activation of {beta}-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against {beta}-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited {beta}-catenin/Tcf signaling efficiently. In addition, the inhibition of {beta}-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant {beta}-catenin gene, whose product is not phosphorylated by GSK3{beta}, indicates that its inhibitory mechanism was related to {beta}-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the {beta}-catenin distribution and the levels of nuclear {beta}-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits {beta}-catenin/Tcf signaling in gastric cancer with unknown mechanisms.

  11. Effects of short-hairpin RNA-inhibited {beta}-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

    SciTech Connect

    Liang, Wenqing; Yang, Chengwei; Qian, Yu; Fu, Qiang

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.

  12. PAK1 interacts with beta-catenin and is required for the regulation of the beta-catenin signalling pathway by gastrins.

    PubMed

    He, Hong; Shulkes, Arthur; Baldwin, Graham S

    2008-10-01

    Beta-catenin regulates cell-cell adhesion by binding to E-cadherin at the cell membrane and, when translocated into the nucleus, mediates signalling by activation of transcription factors such as TCF4. Mutations of the components of the Wnt/beta-catenin pathway are found in many gastrointestinal cancers. Gastrins, including amidated (Gamide) and glycine-extended (Ggly) gastrin(17), stimulate the proliferation of gastrointestinal cancer cells. Gastrins also regulate beta-catenin signalling through multiple pathways which seem to converge on p21-activated kinase 1 (PAK1). In this study, we have investigated the role of PAK1 in the regulation of beta-catenin signalling by gastrins. Here we report for the first time that PAK1 associated with beta-catenin. Both Gamide and Ggly stimulated the phosphorylation and activation of beta-catenin in a PAK1-dependent manner. A kinase-inactive mutant PAK1(K299A) blocked the gastrin-stimulated dissociation of beta-catenin from E-cadherin, translocation of beta-catenin from the cell membrane to the nucleus, and association of beta-catenin with the transcription factor TCF4. The PAK1(K299A) mutant also inhibited the stimulation of the expression of c-myc and cyclin D1, and of cell proliferation and migration, by gastrins. The results indicate that gastrins regulate beta-catenin signalling through a PAK1-dependent pathway. PAK1 seems to be the point of convergence of multiple signalling pathways activated by gastrins.

  13. Polymeric black tea polyphenols inhibit 1,2-dimethylhydrazine induced colorectal carcinogenesis by inhibiting cell proliferation via Wnt/{beta}-catenin pathway

    SciTech Connect

    Patel, Rachana; Ingle, Arvind; Maru, Girish B.

    2008-02-15

    Tea polyphenols like epigallocatechin gallate and theaflavins are established chemopreventive agents for colorectal carcinogenesis. However, studies on evaluating similar chemopreventive properties of thearubigins or polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, are limited. Hence, in the present study we aim to investigate chemopreventive effects along with probable mechanisms of action of PBP extract employing 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in Sprague-Dawley rats as experimental model. The present study suggests that PBPs, like other tea polyphenols, also inhibit DMH-induced colorectal tumorigenesis by decreasing tumor volume and multiplicity. This study also shows that although the pretreatment with PBP extract could induce detoxifying enzymes in hepatic and colorectal tissue, it did not show any additional chemopreventive effects when compared to treatments with PBP extract after initiation with DMH. Mechanistically, PBP extract may inhibit colorectal carcinogenesis by decreasing DMH-induced cell proliferation via Wnt/{beta}-catenin pathway. Treatments with PBP extract showed decreased levels of COX-2, c-MYC and cyclin D1 proteins which aid cell proliferation probably by regulating {beta}-catenin by maintaining expression of APC and decreasing inactivation of GSK3{beta}. DMH-induced activation of MAP kinases such as ERK and JNK was also found to be inhibited by treatments with PBP extract. In conclusion, the protective effects of PBP extract could be attributed to inhibition of DMH-induced cellular proliferation probably through {beta}-catenin regulation.

  14. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular {beta}-catenin protein

    SciTech Connect

    Cho, Munju; Park, Seoyoung; Gwak, Jungsug; Kim, Dong-Eun; Yea, Sung Su; Shin, Jae-Gook; Oh, Sangtaek

    2008-02-29

    The Wnt/{beta}-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/{beta}-catenin signaling pathway. BIM increased {beta}-catenin responsive transcription (CRT) and up-regulated intracellular {beta}-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}) and CAATT enhancer-binding protein {alpha} (C/EBP{alpha}) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of {beta}-catenin protein in 3T3-L1 preadipocyte cells.

  15. The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/{beta}-catenin signaling pathway

    SciTech Connect

    Jeong, Young-Hee; Sekiya, Manami; Hirata, Michiko; Ye, Mingjuan; Yamagishi, Azumi; Lee, Sang-Mi; Kang, Man-Jong; Hosoda, Akemi; Fukumura, Tomoe; Kim, Dong-Ho; Saeki, Shigeru

    2010-02-19

    Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/{beta}-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/{beta}-catenin signaling pathway. The {beta}-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3{beta} phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated {beta}-catenin. Nuclear {beta}-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the {beta}-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/{beta}-catenin signaling pathway.

  16. Rhesus lymphocryptovirus latent membrane protein 2A activates {beta}-catenin signaling and inhibits differentiation in epithelial cells

    SciTech Connect

    Siler, Catherine A.; Raab-Traub, Nancy

    2008-08-01

    Rhesus lymphocryptovirus (LCV) is a {gamma}-herpesvirus closely related to Epstein-Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and {beta}-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3{beta} inactivation and accumulation of {beta}-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of {beta}-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on {beta}-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis.

  17. Inhibition of beta-catenin signaling by Pb leads to incomplete fracture healing

    PubMed Central

    Beier, Eric E; Buckley, Taylor; Yukata, Kiminori; Sheu, Tzong-Jen; O’Keefe, Regis; Zuscik, Michael J; Puzas, J Edward

    2015-01-01

    There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β-catenin levels. Reduction of β-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. β-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β-catenin signaling, suggesting the Wnt/β-catenin as a pivotal pathway in the influence of Pb on fracture repair. PMID:25044211

  18. Inhibition of beta-catenin signaling by Pb leads to incomplete fracture healing.

    PubMed

    Beier, Eric E; Sheu, Tzong-Jen; Buckley, Taylor; Yukata, Kiminori; O'Keefe, Regis; Zuscik, Michael J; Puzas, J Edward

    2014-11-01

    There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β-catenin levels. Reduction of β-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. β-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β-catenin signaling, suggesting the Wnt/β-catenin as a pivotal pathway in the influence of Pb on fracture repair.

  19. The human and mouse sex-determining SRY genes repress the Rspol/beta-catenin signaling.

    PubMed

    Lau, Yun-Fai Chris; Li, Yunmin

    2009-04-01

    The sex-determining region Y (SRY) is the gene on the Y chromosome responsible for switching on male sex determination during mammalian embryogenesis. In its absence, ovaries develop in the embryo. Hence, ovarian determination and differentiation is considered to be a default, or passive, developmental pathway. Recently this classical paradigm of sex determination has been challenged with the discovery of the R-spondin 1 (RSPO1) as an active ovarian determinant. Mutations of RSPO1 cause a female-to-male sex reversal. RSPO1 synergizes with WNT4 in activating an ovarian development in the bipotential gonad via the canonical Wnt signaling. Early studies showed that SRY represses such Wnt signaling, but also generated discrepancies on whether only mouse Sry is capable of inhibiting such Wnt signaling and whether both human and mouse SRY proteins are able to interact with beta-catenin, the intracellular messenger responsible for executing the Wnt signals. Our studies show that both human SRY and mouse Sry are capable of repressing the Rspo1/Wnt/beta-catenin signaling. However, the repression activities vary among different SRY/Sry proteins and paradoxically related to the presence and/or size of an acidic/glutamine-rich domain. The HMG box of human SRY could bind directly to beta-catenin while the mouse Sry binds to beta-catenin via its HMG box and glutamine-rich domain. The results clarify some of the initial discrepancies, and raise the possibility that SRY interacts with beta-catenin in the nucleus and represses the transcriptional activation of the Rspo1/Wnt target genes involved in ovarian determination, thereby switching on testis determination.

  20. E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

    PubMed Central

    1994-01-01

    beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E- cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression. PMID:7806582

  1. Once upon a time there was beta-catenin in cadherin-mediated signalling.

    PubMed

    Gavard, Julie; Mège, René-Marc

    2005-12-01

    beta-Catenin was initially characterized as a protein interacting with the cadherin cytoplasmic tail and regulating cell-cell contacts and actin cytoskeleton interactions. Moreover, the gene coding for the Drosophila orthologue of beta-catenin, armadillo, was independently identified downstream of wingless in the segment-polarity signalling pathway. In fact, beta-catenin/Armadillo turned out to be key mediators of the Wnt/Wingless pathways in vertebrates and invertebrates. beta-Catenin participates in both adhesion and signalling functions in a mutually exclusive manner; bound to cadherins at the plasma membrane or 'unbound' in cytosolic or nuclear complexes. This model had placed beta-catenin at the crossroads between cadherin and Wnt signalling, leading to the dogma of inhibition of beta-catenin signalling by cadherins.

  2. beta-Catenin regulates vascular endothelial growth factor expression in colon cancer.

    PubMed

    Easwaran, Vijay; Lee, Sang H; Inge, Landon; Guo, Lida; Goldbeck, Cheryl; Garrett, Evelyn; Wiesmann, Marion; Garcia, Pablo D; Fuller, John H; Chan, Vivien; Randazzo, Filippo; Gundel, Robert; Warren, Robert S; Escobedo, Jaime; Aukerman, Sharon L; Taylor, Robert N; Fantl, Wendy J

    2003-06-15

    To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.

  3. beta-Catenin promotes self-renewal of skeletal-muscle satellite cells.

    PubMed

    Perez-Ruiz, Ana; Ono, Yusuke; Gnocchi, Viola F; Zammit, Peter S

    2008-05-01

    Satellite cells are the resident stem cells of adult skeletal muscle. As with all stem cells, how the choice between self-renewal or differentiation is controlled is central to understanding their function. Here, we have explored the role of beta-catenin in determining the fate of myogenic satellite cells. Satellite cells express beta-catenin, and expression is maintained as they activate and undergo proliferation. Constitutive retroviral-driven expression of wild-type or stabilised beta-catenin results in more satellite cells expressing Pax7 without any MyoD -- therefore, adopting the self-renewal pathway, with fewer cells undergoing myogenic differentiation. Similarly, preventing the degradation of endogenous beta-catenin by inhibiting GSK3beta activity also results in more Pax7-positive-MyoD-negative (Pax7(+)MyoD(-)) satellite-cell progeny. Consistent with these observations, downregulation of beta-catenin using small interfering RNA (siRNA) reduced the proportion of satellite cells that express Pax7 and augmented myogenic differentiation after mitogen withdrawal. Since a dominant-negative version of beta-catenin had the same effect as silencing beta-catenin using specific siRNA, beta-catenin promotes self-renewal via transcriptional control of target genes. Thus, beta-catenin signalling in proliferating satellite cells directs these cells towards the self-renewal pathway and, so, contributes to the maintenance of this stem-cell pool in adult skeletal muscle.

  4. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway.

    PubMed

    Wang, Yihua; Zhou, Xiaobo; Zhu, Hongxia; Liu, Shuang; Zhou, Cuiqi; Zhang, Guo; Xue, Liyan; Lu, Ning; Quan, Lanping; Bai, Jinfeng; Zhan, Qimin; Xu, Ningzhi

    2005-10-06

    Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or DeltaN TCF4, respectively. Also, we found that EB1 affected the interaction between beta-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of beta-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the beta-catenin/TCF pathway.

  5. Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis.

    PubMed

    Ysebaert, L; Chicanne, G; Demur, C; De Toni, F; Prade-Houdellier, N; Ruidavets, J-B; Mansat-De Mas, V; Rigal-Huguet, F; Laurent, G; Payrastre, B; Manenti, S; Racaud-Sultan, C

    2006-07-01

    Activation of the Wnt/beta-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether beta-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for beta-catenin expression by Western blot. beta-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, beta-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, beta-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in beta-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/beta-catenin pathway only in leukemic cells. Indeed, beta-catenin negative leukemic cells were found to increase beta-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

  6. Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

    PubMed

    Wang, Q; Sun, Z-X; Allgayer, H; Yang, H-S

    2010-01-07

    We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

  7. IKK and (Beta) - Catenin in Breast Cancer

    DTIC Science & Technology

    2004-07-01

    activity is not due to a decrease in total beta-catenin protein levels, however, the dephosphorylated form of beta-catenin within the nucleus...2000. 19. Staal F J, Noort MM, Strous G J, and Clevers HC, Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin. EMBO Rep. 3...catenin is phoshorylated sequentially by casein kinase 1 (CK1) and glycogen synthase kinase 3-P (GSK-313) (4,52); activation of frizzled receptors by Wnt

  8. Cytoplasmic beta-catenin in esophageal cancers.

    PubMed

    Kimura, Y; Shiozaki, H; Doki, Y; Yamamoto, M; Utsunomiya, T; Kawanishi, K; Fukuchi, N; Inoue, M; Tsujinaka, T; Monden, M

    1999-04-20

    beta-Catenin has 2 distinct roles in E-cadherin-mediated cell adhesion and carcinogenesis through APC gene mutation. One occurs at cell-adhesion sites, where cadherins become linked to the actin-based cytoskeleton. The others occur in the cytoplasm and nuclei and are thought to regulate cell transformation. We studied these different beta-catenins and evaluated their significance in carcinogenesis. Fresh surgical specimens were obtained from 22 patients with squamous-cell carcinoma of the esophagus. beta-Catenin in the free soluble fraction and the insoluble fraction was immunoblotted separately. At the same time, its localization was observed by immuno-histochemical techniques. In the normal esophageal epithelium, 91% of beta-catenin was detected in the insoluble fraction and beta-catenin staining occurred at the cell membrane, in co-existence with E-cadherin. In cancerous tissues, the amount of soluble beta-catenin was significantly (about 4-fold) higher than in normal tissues. Also, in cancerous tissues with higher amounts of soluble beta-catenin, immuno-histochemical techniques revealed the presence of beta-catenin in the cytoplasm and nuclei, as well as in the cell membrane. However, in samples with lower amounts of beta-catenin, expression was found only at the cell boundaries. The amount of soluble beta-catenin was not associated with the clinico-pathological grading of the tumors. Our results show that the accumulation of free soluble beta-catenin in the cytoplasm and nuclei frequently occurs during carcinogenesis of the squamous epithelium of the esophagus.

  9. Ibuprofen inhibits activation of nuclear {beta}-catenin in human colon adenomas and induces the phosphorylation of GSK-3{beta}.

    PubMed

    Greenspan, Emily J; Madigan, James P; Boardman, Lisa A; Rosenberg, Daniel W

    2011-01-01

    Nonselective cyclooxygenase (COX) inhibitors target many of the same cancer-associated molecular pathways as COX-2-specific inhibitors. Although these nonsteroidal anti-inflammatory drugs (NSAIDs) are often associated with gastrointestinal toxicity, there is renewed interest in their use as colorectal cancer (CRC) chemopreventive agents due to the adverse side effects associated with long-term use of selective COX-2 inhibitors. In this study, we investigated the effects of long-term use (up to 25 years) of NSAIDs (ibuprofen or aspirin) on adenoma pathology and β-catenin-mediated signaling in sporadic human colon adenomas. Although NSAID use did not impact overall adenoma size or degree of dysplasia, it did cause a significant inhibition of nuclear β-catenin localization, which correlated with suppression of cyclin D1 expression. To further elucidate the effect of these agents in regulating β-catenin, we treated SW480 colon cancer cells with a panel of NSAIDs and determined their effects on β-catenin levels and cellular localization. In agreement with our in vivo results, both S-ibuprofen and aspirin were found to decrease total levels of β-catenin while increasing its phosphorylation. In addition, S-ibuprofen induced both degradation of IκBα and nuclear localization of NF-κB. Despite its nuclear localization, however, the activation of the NF-κB target genes, Bcl-2, survivin, and cyclin D1, was suppressed. This reduction in NF-κB transcriptional activity may be due to increased phosphorylation of GSK-3β following S-ibuprofen treatment. These data suggest that ibuprofen can effectively target both the Wnt/β-catenin and NF-κB pathways, and potentially uncovers a novel mechanism through which NSAIDS may exert their chemopreventive efficacy.

  10. beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex

    PubMed Central

    1996-01-01

    Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a

  11. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

  12. Murrayafoline A attenuates the Wnt/{beta}-catenin pathway by promoting the degradation of intracellular {beta}-catenin proteins

    SciTech Connect

    Choi, Hyuk; Gwak, Jungsug; Cho, Munju; Ryu, Min-Jung; Lee, Jee-Hyun; Kim, Sang Kyum; Kim, Young Ho; Lee, Gye Won; Yun, Mi-Young; Cuong, Nguyen Manh; Shin, Jae-Gook; Song, Gyu-Yong; Oh, Sangtaek

    2010-01-01

    Molecular lesions in Wnt/{beta}-catenin signaling and subsequent up-regulation of {beta}-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3{beta} (GSK-3{beta}), and promoted the degradation of intracellular {beta}-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known {beta}-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

  13. AMP-activated protein kinase (AMPK) cross-talks with canonical Wnt signaling via phosphorylation of {beta}-catenin at Ser 552

    SciTech Connect

    Zhao, Junxing; Yue, Wanfu; Zhu, Mei J.; Sreejayan, Nair; Du, Min

    2010-04-23

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent studies show that AMPK involves in cell differentiation but the underlying mechanism remains undefined. Wingless Int-1 (Wnt)/{beta}-catenin signaling pathway regulates the differentiation of mesenchymal stem cells through enhancing {beta}-catenin/T-cell transcription factor 1 (TCF) mediated transcription. The objective of this study was to determine whether AMPK cross-talks with Wnt/{beta}-catenin signaling through phosphorylation of {beta}-catenin. C3H10T1/2 mesenchymal cells were used. Chemical inhibition of AMPK and the expression of a dominant negative AMPK decreased phosphorylation of {beta}-catenin at Ser 552. The {beta}-catenin/TCF mediated transcription was correlated with AMPK activity. In vitro, pure AMPK phosphorylated {beta}-catenin at Ser 552 and the mutation of Ser 552 to Ala prevented such phosphorylation, which was further confirmed using [{gamma}-{sup 32}P]ATP autoradiography. In conclusion, AMPK phosphorylates {beta}-catenin at Ser 552, which stabilizes {beta}-catenin, enhances {beta}-catenin/TCF mediated transcription, expanding AMPK from regulation of energy metabolism to cell differentiation and development via cross-talking with the Wnt/{beta}-catenin signaling pathway.

  14. SpKrl: a direct target of beta-catenin regulation required for endoderm differentiation in sea urchin embryos.

    PubMed

    Howard, E W; Newman, L A; Oleksyn, D W; Angerer, R C; Angerer, L M

    2001-02-01

    Localization of nuclear beta-catenin initiates specification of vegetal fates in sea urchin embryos. We have identified SpKrl, a gene that is activated upon nuclear entry of beta-catenin. SpKrl is upregulated when nuclear beta-catenin activity is increased with LiCl and downregulated in embryos injected with molecules that inhibit beta-catenin nuclear function. LiCl-mediated SpKrl activation is independent of protein synthesis, indicating that SpKrl is a direct target of beat-catenin and TCF. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, SpKrl mRNA injection rescues some vegetal structures in beta-catenin-deficient embryos. SpKrl negatively regulates expression of the animalizing transcription factor, SpSoxB1. We propose that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities.

  15. Activity-dependent regulation of {beta}-catenin via {epsilon}-cleavage of N-cadherin

    SciTech Connect

    Uemura, Kengo; Kihara, Takeshi; Kuzuya, Akira; Okawa, Katsuya; Nishimoto, Takaaki; Bito, Haruhiko; Ninomiya, Haruaki; Sugimoto, Hachiro; Kinoshita, Ayae . E-mail: akinoshita@hs.med.kyoto-u.ac.jp; Shimohama, Shun

    2006-07-07

    N-cadherin is essential for excitatory synaptic contact in the hippocampus. Presenilin 1 (PS1) is located at sites of synaptic contact, forming a complex with N-cadherin and {beta}-catenin. Here, we report that human N-cadherin is cleaved by PS1/{gamma}-secretase in response to physiological concentration of glutamate (Glu) stimulation, yielding a fragment Ncad/CTF2. The expression of Ncad/CTF2 in neuronal cells led to its translocation to the nucleus, and caused a prominent enhancement of cytoplasmic and nuclear {beta}-catenin levels in a cell-cell contact dependent manner, via following mechanisms: 1, inhibition of {beta}-catenin phosphorylation; 2, transactivation of {beta}-catenin; and 3, inhibition of N-cadherin transcription, and finally enhanced {beta}-catenin nuclear signaling. Since the regulation of cellular {beta}-catenin level is essential for synaptic function, disruption in the cleavage of N-cadherin may be causally linked to the synaptic dysfunction associated with Alzheimer's disease (AD)

  16. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    SciTech Connect

    Sopjani, Mentor; Alesutan, Ioana; Wilmes, Jan; Dermaku-Sopjani, Miribane; Lam, Rebecca S.; Jakupi, Muharrem; Foeller, Michael; Lang, Florian

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  17. Evidence that the beta-catenin nuclear translocation assay allows for measuring presenilin 1 dysfunction.

    PubMed Central

    Van Gassen, G.; De Jonghe, C.; Nishimura, M.; Yu, G.; Kuhn, S.; St George-Hyslop, P.; Van Broeckhoven, C.

    2000-01-01

    BACKGROUND: Mutations in the presenilin (PSEN) genes are responsible for the majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a component of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on beta-catenin and glycogen synthase kinase-3beta(GSK-3beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3beta and PSEN1 were influenced. MATERIALS AND METHODS: Stably transfected human embryonic kidney (HEK) 293 cells overexpressing wild-type (wt) and mutant (mt) PSEN1, treated with and without LiCl, were used to isolate cytoplasmic and nuclear fractions. By Western blot analysis, endogenous beta-catenin levels were examined. By analyzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated the effect of PSEN1 overexpression on beta-catenin stability. Finally, we analyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3beta by co-immunoprecipitation experiments. RESULTS: We report reduced nuclear translocation of beta-catenin in cells stably expressing I143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of beta-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid beta(Abeta). Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of beta-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3beta was observed when GSK3beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3beta. CONCLUSION: Our results further establish the nuclear

  18. Wnt/{beta}-catenin signaling regulates cancer stem cells in lung cancer A549 cells

    SciTech Connect

    Teng, Ying; Wang, Xiuwen; Wang, Yawei; Ma, Daoxin

    2010-02-12

    Wnt/{beta}-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that {beta}-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of {beta}-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of {beta}-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/{beta}-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.

  19. Wnt-3A/beta-catenin signaling induces transcription from the LEF-1 promoter.

    PubMed

    Filali, Mohammed; Cheng, Ningli; Abbott, Duane; Leontiev, Vladimir; Engelhardt, John F

    2002-09-06

    Members of the Wnt family of secreted molecules have been established as key factors in determining cell fate and morphogenic signaling. It has long been recognized that Wnt induces morphogenic signaling through the Tcf/LEF-1 cascade by regulating free intracellular levels of beta-catenin, a co-factor for Tcf/LEF-1 transcription factors. In the present study, we have demonstrated that Wnt-3A can also directly induce transcription from the LEF-1 promoter. This induction was dependent on glycogen synthase kinase 3beta inactivation, a rise in free intracellular beta-catenin, and a short 110-bp Wnt-responsive element (WRE) in the LEF-1 promoter. Linear and internal deletion of this WRE led to a dramatic increase in constitutive LEF-1 promoter activity and loss of Wnt-3A responsiveness. In isolation, the 110-bp WRE conferred context-independent Wnt-3A or beta-catenin(S37A) responsiveness to a heterologous SV40 promoter. Studies expressing dominant active and negative forms of LEF-1, beta-catenin, GSK-3beta, and beta-catenin/LEF-1 fusions suggest that Wnt-3A activates the LEF-1 promoter through a beta-catenin-dependent and LEF-1-independent process. Wnt-3A expression also induced multiple changes in the binding of factors to the WRE and suggests that regulatory mechanisms may involve modulation of a multiprotein complex. In summary, these results provide evidence for transcriptional regulation of the LEF-1 promoter by Wnt and enhance the mechanistic understanding of Wnt/beta-catenin signaling in the regulation of LEF-1-dependent developmental processes.

  20. Smad6 promotes neuronal differentiation in the intermediate zone of the dorsal neural tube by inhibition of the Wnt/beta-catenin pathway.

    PubMed

    Xie, Zhihui; Chen, Yongfeng; Li, Zhenfei; Bai, Ge; Zhu, Yue; Yan, Rui; Tan, Fangzhi; Chen, Ye-Guang; Guillemot, Francois; Li, Lin; Jing, Naihe

    2011-07-19

    Proliferation of the neural/neuronal progenitor cells (NPCs) at the ventricular zone of the dorsal spinal cord requires the stimuli of Wnt and bone morphogenic protein (BMP). However, how these two signaling pathways are regulated to initiate differentiation in the NPCs as they enter the intermediate zone is not known. Here, we show that Smad6, a negative regulator of BMP signaling, is expressed in the intermediate zone of the chick dorsal spinal cord. Knockdown experiments show that Smad6 is required for promoting NPCs to exit the cell cycle and differentiate into neurons. Although we find that Smad6 inhibits BMP signaling, as expected, we also find that Smad6 unexpectedly inhibits the Wnt/β-catenin pathway. The inhibition of the Wnt/β-catenin pathway by Smad6 is independent of its effect on the BMP pathway. Rather, Smad6 through its N-terminal domain and link region enhances the interaction of C-terminal binding protein with the β-catenin/T cell factor (TCF) complex and the TCF-binding element to inhibit β-catenin-mediated transcriptional activation. Our study provides evidence that transition of NPCs from a proliferative state to a differentiating state is controlled by the dual inhibitory role of Smad6 to both BMP and Wnt signaling at the level of transcription.

  1. Interactions between PPAR Gamma and the Canonical Wnt/Beta-Catenin Pathway in Type 2 Diabetes and Colon Cancer

    PubMed Central

    Claes, Victor

    2017-01-01

    In both colon cancer and type 2 diabetes, metabolic changes induced by upregulation of the Wnt/beta-catenin signaling and downregulation of peroxisome proliferator-activated receptor gamma (PPAR gamma) may help account for the frequent association of these two diseases. In both diseases, PPAR gamma is downregulated while the canonical Wnt/beta-catenin pathway is upregulated. In colon cancer, upregulation of the canonical Wnt system induces activation of pyruvate dehydrogenase kinase and deactivation of the pyruvate dehydrogenase complex. As a result, a large part of cytosolic pyruvate is converted into lactate through activation of lactate dehydrogenase. Lactate is extruded out of the cell by means of activation of monocarboxylate lactate transporter-1. This phenomenon is called Warburg effect. PPAR gamma agonists induce beta-catenin inhibition, while inhibition of the canonical Wnt/beta-catenin pathway activates PPAR gamma. PMID:28298922

  2. Isoreserpine promotes {beta}-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells

    SciTech Connect

    Gwak, Jungsug; Song, Taeyun; Song, Jie-Young; Yun, Yeon-Sook; Choi, Il-Whan; Jeong, Yongsu; Shin, Jae-Gook; Oh, Sangtaek

    2009-09-25

    Aberrant accumulation of intracellular {beta}-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular {beta}-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular {beta}-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of {beta}-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

  3. Beta-catenin--a supporting role in the skeleton.

    PubMed

    Case, Natasha; Rubin, Janet

    2010-06-01

    In the last 5 years a role for beta-catenin in the skeleton has been cemented. Beginning with mutations in the Lrp5 receptor that control beta-catenin canonical downstream signals, and progressing to transgenic models with bone-specific alteration of beta-catenin, research has shown that beta-catenin is required for normal bone development. A cell critical to bone in which beta-catenin activity determines function is the marrow-derived mesenchymal stem cell (MSC), where sustained beta-catenin prevents its distribution into adipogenic lineage. beta-Catenin actions are less well understood in mature osteoblasts: while beta-catenin contributes to control of osteoclastic bone resorption via alteration of the osteoprotegerin/RANKL ratio, a specific regulatory role during osteoblast bone synthesis has not yet been determined. The proven ability of mechanical factors to prevent beta-catenin degradation and induce nuclear translocation through Lrp-independent mechanisms suggests processes by which exercise might modulate bone mass via control of lineage allocation, in particular, by preventing precursor distribution into the adipocyte pool. Effects resulting from mechanical activation of beta-catenin in mature osteoblasts and osteocytes likely modulate bone resorption, but whether beta-catenin is involved in osteoblast synthetic function remains to be proven for both mechanical and soluble mediators. As beta-catenin appears to support the downstream effects of multiple osteogenic factors, studies clarifying when and where beta-catenin effects occur will be relevant for translational approaches aimed at preventing bone loss and terminal adipogenic conversion.

  4. Micro RNA-214 contributes to proteasome independent downregulation of beta catenin in Huntington's disease knock-in striatal cell model STHdhQ111/Q111.

    PubMed

    Ghatak, Supratim; Raha, Sanghamitra

    2015-04-10

    Role of beta catenin in Huntington's disease (HD) is not clear. Previous studies on HD reported varied levels of beta catenin. In the present study we showed that beta catenin is post transcriptionally down-regulated in mutant huntingtin knock-in cell model STHdhQ111/Q111. This in turn leads to decreased level of wnt/beta catenin responsive genes. We observed that Gsk3beta or Gsk3beta (phospho Ser 9) is unaltered in HD and this down-regulation of beta catenin is independent of proteasomal degradation. Finally, we showed that the overexpression of miR-214 leads to the down-regulation of beta catenin at protein level only and reduces its transcriptional activity. We concluded that, miR-214 contributes to the processes that result in proteasome independent post transcriptional down-regulation of beta catenin in STHdhQ111/Q111, probably through inhibition of protein synthesis from beta catenin mRNA.

  5. Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction

    PubMed Central

    Wei, Lei; Mo, Mingshu; Feng, Junmin; Sun, Congcong; Xiao, Yousheng; Luo, Qin; Li, Shaomin; Yang, Xinling; Xu, Pingyi

    2016-01-01

    Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta. Increasing evidence showed that Wnt/β-catenin pathway and the orphan nuclear receptor Nurr1 play crucial roles in the survival and functional maintenance of DA neurons in the midbrain and GSK-3β antagonists LiCl and SB216763 were used to activate Wnt/β-catenin pathway experimentally. However, the detail mechanism underlying the neuroprotection against apoptosis on DA neuron is still unclear and the interaction between Wnt/β-catenin and Nurr1 remains undisclosed. In this study, using cell biological assay we investigated the function of Wnt/β-catenin and its crosstalk with Nurr1 on the course of PC12 cell degeneration in vitro. Our data showed that PC12 cell viability was inhibited by rotenone, but attenuated by GSK-3β antagonists LiCl or SB216763. The activity of Wnt/β-catenin pathway was deregulated on exposure of rotenone in a concentration-dependent manner. After the interference of β-catenin with siRNA, LiCl or SB216763 failed to protect PC12 cells from apoptosis by the rotenone toxicity. Our data confirmed that Wnt/β-catenin signaling activated by LiCl or SB216763 enhanced Nurr1 expression to 2.75 ± 0.55 and 4.06 ± 0.41 folds respectively compared with control detected by real-time PCR and the interaction of β-catenin with Nurr1 was identified by co-immunoprecipitate analysis. In conclusion, the data suggested that Wnt/β-catenin and Nurr1 are crucial factors in the survival of DA neurons, and the activation of Wnt/β-catenin pathway exerts protective effects on DA neurons partly by mean of a co-active pattern with Nurr1. This finding may shed a light on the potential treatment of Parkinson disease. PMID:27045591

  6. Expression of beta-catenin is regulated by PI-3 kinase and sodium butyrate in colorectal cancer cells.

    PubMed

    Turecková, Jolana; Kucerová, Dana; Vojtechová, Martina; Sloncová, Eva; Tuhácková, Zdena

    2006-01-01

    beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.

  7. Beta-catenin expression in psoriasis

    PubMed Central

    El-wahed Gaber, Mohamed Abd; El-Halim Kandil, Mona Abd; El-Farargy, Shawki Mahmoud; Galbet, Doaa Abd Elmoniem

    2015-01-01

    Background: Psoriasis is a common inflammatory skin disease characterized by abnormal keratinocyte proliferation and differentiation. Beta-catenin participates in intercellular adhesion. Catenins are proteins found in complexes with cadherin cell adhesion molecules of cells. The role of catenin in regulating keratinocyte stem cell differentiation and hair follicle morphogenesis has been extensively reported. Aims and Objectives: is to study β-catenin expression in lesional and non-lesional psoriatic skin to throw light upon its possible role in the pathogenesis of psoriasis. Materials and Methods: Biopsies were taken from 20 patients with psoriasis vulgaris and from 10 normal controls. The distribution of Beta catenin was investigated using polycolonal rabbits B-catenin antibody-1 by immunohistochemical method. Results: In this study membranous β-catenin expression was significantly demonstrated in the control group then the non-lesional areas in comparison to the lesional areas (P < 0.001). Nuclear β-catenin staining expression was significantly more demonstrated in lesional and non-lesional areas in comparison to the control cases (P < 0.001). Conclusions: The down regulation of membranous β-catenin expression in lesional psoriatic skin might reflect a useful phenotypic marker of hyperprolifration of keratinocytes in psoriasis. Moreover, the mild down regulation of membranous β-catenin expression in non lesional psoriatic skin may provide clues about incipient structural abnormalities in the pathogenesis of psoriasis, providing an early diagnostic indicator for evolution to a generalized form of the disease. Nuclear β-catenin expression was not found in the control group but was demonstrated in lesional and moderately in non-lesional reflecting its role in kerationcyte proliferation. PMID:25657910

  8. Natural derivatives of curcumin attenuate the Wnt/{beta}-catenin pathway through down-regulation of the transcriptional coactivator p300

    SciTech Connect

    Ryu, Min-Jung; Cho, Munju; Song, Jie-Young; Yun, Yeon-Sook; Choi, Il-Whan; Kim, Dong-Eun; Park, Byeoung-Soo; Oh, Sangtaek

    2008-12-26

    Curcumin, a component of turmeric (Curcuma longa), has been reported to suppress {beta}-catenin response transcription (CRT), which is aberrantly activated in colorectal cancer. However, the effects of its natural analogs (demethoxycurcumin [DMC] and bisdemethoxycurcumin [BDMC]) and metabolite (tetrahydrocurcumin [THC]) on the Wnt/{beta}-catenin pathway have not been investigated. Here, we show that DMC and BDMC suppressed CRT that was activated by Wnt3a conditioned-medium (Wnt3a-CM) without altering the level of intracellular {beta}-catenin, and inhibited the growth of various colon cancer cells, with comparable potency to curcumin. Additionally, DMC and BDMC down-regulated p300, which is a positive regulator of the Wnt/{beta}-catenin pathway. Notably, THC also inhibited CRT and cell proliferation, but to a much lesser degree than curcumin, DMC, or BDMC, indicating that the conjugated bonds in the central seven-carbon chain of curcuminoids are essential for the inhibition of Wnt/{beta}-catenin pathway and the anti-proliferative activity of curcuminoids. Thus, our findings suggest that curcumin derivatives inhibit the Wnt/{beta}-catenin pathway by decreasing the amount of the transcriptional coactivator p300.

  9. Concurrent Transient Activation of Wnt/{beta}-Catenin Pathway Prevents Radiation Damage to Salivary Glands

    SciTech Connect

    Hai Bo; Yang Zhenhua; Shangguan Lei; Zhao Yanqiu; Boyer, Arthur; Liu, Fei

    2012-05-01

    Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/{beta}-catenin signaling in preventing radiation damage to salivary glands in a preclinical model. Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after, or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated. Results: Radiation damage did not significantly affect activity of Wnt/{beta}-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/{beta}-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window. Conclusion: These results suggest that concurrent transient activation of the Wnt/{beta}-catenin pathway could prevent radiation-induced salivary gland dysfunction.

  10. Alzheimer Disease: Crosstalk between the Canonical Wnt/Beta-Catenin Pathway and PPARs Alpha and Gamma

    PubMed Central

    Vallée, Alexandre; Lecarpentier, Yves

    2016-01-01

    The molecular mechanisms underlying the pathophysiology of Alzheimer's disease (AD) are still not fully understood. In AD, Wnt/beta-catenin signaling has been shown to be downregulated while the peroxisome proliferator-activated receptor (PPAR) gamma (mARN and protein) is upregulated. Certain neurodegenerative diseases share the same Wnt/beta-catenin/PPAR gamma profile, such as bipolar disorder and schizophrenia. Conversely, other NDs share an opposite profile, such as amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, multiple sclerosis, and Friedreich's ataxia. AD is characterized by the deposition of extracellular Abeta plaques and the formation of intracellular neurofibrillary tangles in the central nervous system (CNS). Activation of Wnt signaling or inhibition of both glycogen synthase kinase-3beta and Dickkopf 1, two key negative regulators of the canonical Wnt pathway, are able to protect against Abeta neurotoxicity and to ameliorate cognitive performance in AD patients. Although PPAR gamma is upregulated in AD patients, and despite the fact that it has been shown that the PPAR gamma and Wnt/beta catenin pathway systems work in an opposite manner, PPAR gamma agonists diminish learning and memory deficits, decrease Abeta activation of microglia, and prevent hippocampal and cortical neurons from dying. These beneficial effects observed in AD transgenic mice and patients might be partially due to the anti-inflammatory properties of PPAR gamma agonists. Moreover, activation of PPAR alpha upregulates transcription of the alpha-secretase gene and represents a new therapeutic treatment for AD. This review focuses largely on the behavior of two opposing pathways in AD, namely Wnt/beta-catenin signaling and PPAR gamma. It is hoped that this approach may help to develop novel AD therapeutic strategies integrating PPAR alpha signaling. PMID:27807401

  11. Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning.

    PubMed

    Schneider, Igor; Houston, Douglas W; Rebagliati, Michael R; Slusarski, Diane C

    2008-01-01

    Establishment of the left-right axis is essential for normal organ morphogenesis and function. Ca(2+) signaling and cilia function in the zebrafish Kuppfer's Vesicle (KV) have been implicated in laterality. Here we describe an endogenous Ca(2+) release event in the region of the KV precursors (dorsal forerunner cells, DFCs), prior to KV and cilia formation. Manipulation of Ca(2+) release to disrupt this early flux does not impact early DFC specification, but results in altered DFC migration or cohesion in the tailbud at somite stages. This leads to disruption of KV formation followed by bilateral expression of asymmetrical genes, and randomized organ laterality. We identify beta-catenin inhibition as a Ca(2+)-signaling target and demonstrate that localized loss of Ca(2+) within the DFC region or DFC-specific activation of beta-catenin is sufficient to alter laterality in zebrafish. We identify a previously unknown DFC-like cell population in Xenopus and demonstrate a similar Ca(2+)-sensitive stage. As in zebrafish, manipulation of Ca(2+) release results in ectopic nuclear beta-catenin and altered laterality. Overall, our data support a conserved early Ca(2+) requirement in DFC-like cell function in zebrafish and Xenopus.

  12. Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin.

    PubMed

    Goodall, Jane; Martinozzi, Silvia; Dexter, Timothy J; Champeval, Delphine; Carreira, Suzanne; Larue, Lionel; Goding, Colin R

    2004-04-01

    Constitutive activation of the Wnt/beta-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/beta-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing beta-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.

  13. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    SciTech Connect

    Miclea, Razvan L.; Horst, Geertje van der; Robanus-Maandag, Els C.; Loewik, Clemens W.G.M.; Oostdijk, Wilma; Wit, Jan M.; Karperien, Marcel

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  14. PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin.

    PubMed

    Ilan, N; Mahooti, S; Rimm, D L; Madri, J A

    1999-09-01

    Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

  15. WNT10B functional dualism: beta-catenin/Tcf-dependent growth promotion or independent suppression with deregulated expression in cancer.

    PubMed

    Yoshikawa, Hirohide; Matsubara, Kenichi; Zhou, Xiaoling; Okamura, Shu; Kubo, Takahiko; Murase, Yaeko; Shikauchi, Yuko; Esteller, Manel; Herman, James G; Wei Wang, Xin; Harris, Curtis C

    2007-11-01

    We found aberrant DNA methylation of the WNT10B promoter region in 46% of primary hepatocellular carcinoma (HCC) and 15% of colon cancer samples. Three of 10 HCC and one of two colon cancer cell lines demonstrated low or no expression, and 5-aza-2'deoxycytidine reactivated WNT10B expression with the induction of demethylation, indicating that WNT10B is silenced by DNA methylation in some cancers, whereas WNT10B expression is up-regulated in seven of the 10 HCC cell lines and a colon cancer cell line. These results indicate that WNT10B can be deregulated by either overexpression or silencing in cancer. We found that WNT10B up-regulated beta-catenin/Tcf activity. However, WNT10B-overexpressing cells demonstrated a reduced growth rate and anchorage-independent growth that is independent of the beta-catenin/Tcf activation, because mutant beta-catenin-transduced cells did not suppress growth, and dominant-negative hTcf-4 failed to alleviate the growth suppression by WNT10B. Although WNT10B expression alone inhibits cell growth, it acts synergistically with the fibroblast growth factor (FGF) to stimulate cell growth. WNT10B is bifunctional, one function of which is involved in beta-catenin/Tcf activation, and the other function is related to the down-regulation of cell growth through a different mechanism. We suggest that FGF switches WNT10B from a negative to a positive cell growth regulator.

  16. MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

    SciTech Connect

    Sun, Jian-Yong; Huang, Yi; Li, Ji-Peng; Zhang, Xiang; Wang, Lei; Meng, Yan-Ling; Yan, Bo; Bian, Yong-Qian; Zhao, Jing; Wang, Wei-Zhong; and others

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colon cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.

  17. Lithium and neuropsychiatric therapeutics: neuroplasticity via glycogen synthase kinase-3beta, beta-catenin, and neurotrophin cascades.

    PubMed

    Wada, Akihiko

    2009-05-01

    Mood disorders are not merely attributed to the functional defect of neurotransmission, but also are due to the structural impairment of neuroplasticity. Chronic stress decreases neurotrophin levels, precipitating or exacerbating depression; conversely, antidepressants increase expression of various neurotrophins (e.g., brain-derived neurotrophic factor and vascular endothelial growth factor), thereby blocking or reversing structural and functional pathologies via promoting neurogenesis. Since the worldwide approval of lithium therapy in 1970, lithium has been used for its anti-manic, antidepressant, and anti-suicidal effects, yet the therapeutic mechanisms at the cellular level remain not-fully defined. During the last five years, multiple lines of evidence have shown that the mood stabilization and neurogenesis by lithium are due to the lithium-induced inhibition of glycogen synthase kinase-3beta (GSK-3beta), allowing accumulation of beta-catenin and beta-catenin-dependent gene transcriptional events. Altered levels of GSK-3beta and beta-catenin are associated with various neuropsychiatric and neurodegenerative diseases, while various classical neuropsychiatric drugs inhibit GSK-3beta and up-regulate beta-catenin expression. In addition, evidence has emerged that insulin-like growth factor-I enhances antidepression, anti-anxiety, memory, neurogenesis, and angiogenesis; antidepressants up-regulate expression of insulin-like growth factor-I, while insulin-like growth factor-I up-regulates brain-derived neurotrophic factor expression and its receptor TrkB level, as well as brain-derived neurotrophic factor-induced synaptic protein levels. More importantly, physical exercise and healthy diet raise transport of peripheral circulating insulin-like growth factor I into the brain, reinforcing the expression of neurotrophins (e.g., brain-derived neurotrophic factor) and the strength of cell survival signalings (e.g., phosphoinositide 3-kinase / Akt / GSK-3beta pathway

  18. O-GlcNAc-glycosylation of {beta}-catenin regulates its nuclear localization and transcriptional activity

    SciTech Connect

    Sayat, Ria; Leber, Brian; Grubac, Vanja; Wiltshire, Lesley; Persad, Sujata

    2008-09-10

    {beta}-catenin plays a role in intracellular adhesion and regulating gene expression. The latter role is associated with its oncogenic properties. Phosphorylation of {beta}-catenin controls its intracellular expression but mechanism/s that regulates the nuclear localization of {beta}-catenin is unknown. We demonstrate that O-GlcNAc glycosylation (O-GlcNAcylation) of {beta}-catenin negatively regulates its levels in the nucleus. We show that normal prostate cells (PNT1A) have significantly higher amounts of O-GlcNAcylated {beta}-catenin compared to prostate cancer (CaP) cells. The total nuclear levels of {beta}-catenin are higher in the CaP cells than PNT1A but only a minimal fraction of the nuclear {beta}-catenin in the CaP cells are O-GlcNAcylated. Increasing the levels of O-GlcNAcylated {beta}-catenin in the CaP cells with PUGNAc (O- (2-acetamido-2-deoxy-D-gluco-pyranosylidene) amino-N-phenylcarbamate) treatment is associated with a progressive decrease in the levels of {beta}-catenin in the nucleus. TOPFlash reporter assay and mRNA expressions of {beta}-catenin's target genes indicate that O-GlcNAcylation of {beta}-catenin results in a decrease in its transcriptional activity. We define a novel modification of {beta}-catenin that regulates its nuclear localization and transcriptional function.

  19. Convergence of 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A and glycogen synthase kinase-3beta/beta-catenin signaling in corpus luteum progesterone synthesis.

    PubMed

    Roy, Lynn; McDonald, Claudia A; Jiang, Chao; Maroni, Dulce; Zeleznik, Anthony J; Wyatt, Todd A; Hou, Xiaoying; Davis, John S

    2009-11-01

    Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3beta and beta-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3beta at N-terminal Ser9 causing its inactivation and resulted in the accumulation of beta-catenin. Overexpression of N-terminal truncated beta-catenin (Delta90 beta-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3beta (GSK-S9A) reduced beta-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of beta-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3beta/beta-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

  20. Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers

    PubMed Central

    De, Pradip; Carlson, Jennifer H.; Wu, Hui; Marcus, Adam; Leyland-Jones, Brian; Dey, Nandini

    2016-01-01

    Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronection-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship

  1. Teaching resource. Beta-catenin signaling and axis specification.

    PubMed

    Moon, Randall T

    2004-06-29

    This animation provides a representation of the beta-catenin signaling pathway in response to fertilization and the process of axis specification that occurs early in development. The process shown is based on analysis of embryos of the amphibian Xenopus. This animation would be useful in illustrating events that occur early in embryogenesis and how embryos become polarized as a consequence of localized signaling processes.

  2. Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/{beta}-catenin pathway in human colorectal adenocarcinoma DLD1 cells

    SciTech Connect

    Zhang Zhuo; Wang Xin; Cheng Senping; Sun Lijuan; Son, Young-Ok; Yao Hua; Li Wenqi; Budhraja, Amit; Li Li; Shelton, Brent J.; Tucker, Thomas; Arnold, Susanne M.; Shi Xianglin

    2011-10-15

    Long term exposure to arsenic can increase incidence of human cancers, such as skin, lung, and colon rectum. The mechanism of arsenic induced carcinogenesis is still unclear. It is generally believed that reactive oxygen species (ROS) may play an important role in this process. In the present study, we investigate the possible linkage between ROS, {beta}-catenin and arsenic induced transformation and tumorigenesis in human colorectal adenocarcinoma cell line, DLD1 cells. Our results show that arsenic was able to activate p47{sup phox} and p67{sup phox}, two key proteins for activation of NADPH oxidase. Arsenic was also able to generate ROS in DLD1 cells. Arsenic increased {beta}-catenin expression level and its promoter activity. ROS played a major role in arsenic-induced {beta}-catenin activation. Treatment of DLD1 cells by arsenic enhanced both transformation and tumorigenesis of these cells. The tumor volumes of arsenic treated group were much larger than those without arsenic treatment. Addition of either superoxide dismutase (SOD) or catalase reduced arsenic induced cell transformation and tumor formation. The results indicate that ROS are involved in arsenic induced cell transformation and tumor formation possible through Wnt/{beta}-catenin pathway in human colorectal adenocarcinoma cell line DLD1 cells. - Highlights: > Arsenic activates NADPH oxidase and increases reactive oxygen species generation in DLD1 cells. > Arsenic increases {beta}-catenin expression. > Inhibition of ROS induced by arsenic reduce {beta}-catenin expression. > Arsenic increases cell transformation in DLD1 cells and tumorigenesis in nude mice. > Blockage of ROS decrease cell transformation and tumorigenesis induced by arsenic.

  3. Cables links Robo-bound Abl kinase to N-cadherin-bound beta-catenin to mediate Slit-induced modulation of adhesion and transcription.

    PubMed

    Rhee, Jinseol; Buchan, Tim; Zukerberg, Lawrence; Lilien, Jack; Balsamo, Janne

    2007-08-01

    Binding of the secreted axon guidance cue Slit to its Robo receptor results in inactivation of the neural, calcium-dependent cell-cell adhesion molecule N-cadherin, providing a rapid epigenetic mechanism for integrating guidance and adhesion information. This requires the formation of a multimolecular complex containing Robo, Abl tyrosine kinase and N-cadherin. Here we show that on binding of Slit to Robo, the adaptor protein Cables is recruited to Robo-associated Abl and forms a multimeric complex by binding directly to N-cadherin-associated beta-catenin. Complex formation results in Abl-mediated phosphorylation of beta-catenin on tyrosine 489, leading to a decrease in its affinity for N-cadherin, loss of N-cadherin function, and targeting of phospho-Y489-beta-catenin to the nucleus. Nuclear beta-catenin combines with the transcription factor Tcf/Lef and activates transcription. Thus, Slit-induced formation of the Robo-N-cadherin complex results in a rapid loss of cadherin-mediated adhesion and has more lasting effects on gene transcription.

  4. Flavonoids of Herba Epimedii regulate osteogenesis of human mesenchymal stem cells through BMP and Wnt/beta-catenin signaling pathway.

    PubMed

    Zhang, Jin-fang; Li, Guo; Chan, Chu-yan; Meng, Chun-ling; Lin, Marie Chia-mi; Chen, Yang-chao; He, Ming-liang; Leung, Ping-chung; Kung, Hsiang-fu

    2010-01-15

    Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, beta-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/beta-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/beta-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.

  5. Teaching resource. Canonical Wnt/beta-catenin signaling.

    PubMed

    Moon, Randall T

    2004-06-29

    This animation provides an interactive presentation of the Wnt signaling pathway as it may occur in multiple cell types. This animation would be useful in teaching developmental biology, immunology, and cell signaling courses. Activation of Wnt pathways can modulate cell proliferation, cell survival, cell behavior, and cell fate. In the basal, unstimulated state in the absence of ligand, there is a constitutively active kinase, which phosphorylates target proteins, resulting in their degradation. Thus, the presence of the ligand Wnt inactivates the kinase allowing accumulation of beta-catenin, which then translocates to the nucleus and acts as a transcriptional regulator.

  6. Gamma-Secretase Inhibitor IX (GSI) Impairs Concomitant Activation of Notch and wnt-beta-catenin Pathways in CD44(+) Gastric Cancer Stem Cells.

    PubMed

    Barat, Samarpita; Chen, Xi; Cuong Bui, Khac; Bozko, Przemyslaw; Götze, Julian; Christgen, Matthias; Krech, Till; Malek, Nisar P; Plentz, Ruben R

    2017-02-03

    Cancer stem cells (CSC) are associated with tumor resistance and are characterized in gastric cancer (GC). Studies have indicated that Notch and wnt-beta-catenin pathways are crucial for CSC development. Using CD44(+) CSCs, we investigated the role of these pathways in GC carcinogenesis. We performed cell proliferation, wound healing, invasion, tumorsphere, and apoptosis assays. Immunoblot analysis of downstream signaling targets of Notch and wnt-beta-catenin were tested after gamma-secretase inhibitor IX (GSI) treatment. Immunohistochemistry, immunofluorescence, and Fluorescence activated cell sorting (FACS) were used to determine CD44 and Hairy enhancer of split-1 (Hes1) expression in human GC tissues. CD44(+) CSCs were subcutaneously injected into NMR-nu/nu mice and treated with vehicle or GSI. GC patients with expression of CD44 and Hes1 showed overall reduced survival. CD44(+) CSCs showed high expression of Hes1. GSI treatment showed effective inhibition of cell proliferation, migration, invasion, tumor sphere formation of CD44(+) CSCs, and induced apoptosis. Importanly, Notch1 was found to be important in mediating a crosstalk between Notch and wnt-beta-catenin in CD44(+) CSCs. Our study highlights a crosstalk between Notch and wnt-beta-catenin in gastric CD44(+) CSCs. Expression of CD44 and Hes1 is associated with patient overall survival. GSI could be an alternative drug to treat GC. © Stem Cells Translational Medicine 2017.

  7. Amide derivatives of ethacrynic acid: synthesis and evaluation as antagonists of Wnt/beta-catenin signaling and CLL cell survival.

    PubMed

    Jin, Guangyi; Lu, Desheng; Yao, Shiyin; Wu, Christina C N; Liu, Jerry X; Carson, Dennis A; Cottam, Howard B

    2009-02-01

    A series of amides of ethacrynic acid was prepared and evaluated for their ability to inhibit Wnt signaling and decrease the survival of CLL cells. Several of the most potent derivatives were active in the low micromolar range. Reduction of the alpha,beta-unsaturated carbon-carbon double bond of EA abrogated both the inhibition of Wnt signaling as well as the decrease in CLL survival. Preliminary mechanism of action studies suggest that these derivatives covalently modify sulfhydryl groups present on transcription factors important for Wnt/beta-catenin signaling.

  8. Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

    SciTech Connect

    Scherbakov, Alexander M.; Stefanova, Lidia B.; Sorokin, Danila V.; Semina, Svetlana E.; Berstein, Lev M.; Krasil’nikov, Mikhail A.

    2013-12-10

    The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O{sub 2} atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling. Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well

  9. E-cadherin is required for caveolin-1-mediated down-regulation of the inhibitor of apoptosis protein survivin via reduced beta-catenin-Tcf/Lef-dependent transcription.

    PubMed

    Torres, Vicente A; Tapia, Julio C; Rodriguez, Diego A; Lladser, Alvaro; Arredondo, Cristian; Leyton, Lisette; Quest, Andrew F G

    2007-11-01

    Caveolin-1 reportedly acts as a tumor suppressor and promotes events associated with tumor progression, including metastasis. The molecular mechanisms underlying such radical differences in function are not understood. Recently, we showed that caveolin-1 inhibits expression of the inhibitor of apoptosis protein survivin via a transcriptional mechanism involving the beta-catenin-Tcf/Lef pathway. Surprisingly, while caveolin-1 expression decreased survivin mRNA and protein levels in HT29(ATCC) human colon cancer cells, this was not the case in metastatic HT29(US) cells. Survivin down-regulation was paralleled by coimmunoprecipitation and colocalization of caveolin-1 with beta-catenin in HT29(ATCC) but not HT29(US) cells. Unlike HT29(ATCC) cells, HT29(US) cells expressed small amounts of E-cadherin that accumulated in intracellular patches rather than at the cell surface. Re-expression of E-cadherin in HT29(US) cells restored the ability of caveolin-1 to down-regulate beta-catenin-Tcf/Lef-dependent transcription and survivin expression, as seen in HT29(ATCC) cells. In addition, coimmunoprecipitation and colocalization between caveolin-1 and beta-catenin increased upon E-cadherin expression in HT29(US) cells. In human embryonic kidney HEK293T and HT29(US) cells, caveolin-1 and E-cadherin cooperated in suppressing beta-catenin-Tcf/Lef-dependent transcription as well as survivin expression. Finally, mouse melanoma B16-F10 cells, another metastatic cell model with low endogenous caveolin-1 and E-cadherin levels, were characterized. In these cells, caveolin-1-mediated down-regulation of survivin in the presence of E-cadherin coincided with increased apoptosis. Thus, the absence of E-cadherin severely compromises the ability of caveolin-1 to develop activities potentially relevant to its role as a tumor suppressor.

  10. Wnt5a is required for endothelial differentiation of embryonic stem cells and vascularization via pathways involving both Wnt/beta-catenin and protein kinase Calpha.

    PubMed

    Yang, Dong-Hwa; Yoon, Ju-Young; Lee, Soung-Hoon; Bryja, Vitezslav; Andersson, Emma R; Arenas, Ernest; Kwon, Young-Guen; Choi, Kang-Yell

    2009-02-13

    In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a(-/-) mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Involvement of both beta-catenin and protein kinase (PK)Calpha pathways in endothelial differentiation of mES cells requiring Wnt5a was indicated by activation of both beta-catenin and PKCalpha in Wnt5a(+/-) but not in Wnt5a(-/-) mES cells. We also found that beta-catenin or PKCalpha knockdowns inhibited the Wnt5a-induced endothelial differentiation of ES cells. Moreover, the lack of endothelial differentiation of Wnt5a(-/-) mES cells was rescued only by transfection of both beta-catenin and PKCalpha, indicating that both genes are required for Wnt5a-mediated endothelial differentiation. Wnt5a was also found to be essential for the differentiation of mES cells into immature endothelial progenitor cells, which are known to play a role in repair of damaged endothelium. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a(-/-) mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/beta-catenin and PKC signaling pathways and regulates embryonic vascular development.

  11. Excess beta-catenin promotes accumulation of transcriptionally active p53.

    PubMed Central

    Damalas, A; Ben-Ze'ev, A; Simcha, I; Shtutman, M; Leal, J F; Zhurinsky, J; Geiger, B; Oren, M

    1999-01-01

    beta-catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals. Deregulated beta-catenin protein expression, due to mutations in the beta-catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein. Increased expression of beta-catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor. To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess beta-catenin on p53. We report here that overexpression of beta-catenin results in accumulation of p53, apparently through interference with its proteolytic degradation. This effect involves both Mdm2-dependent and -independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells. Increased p53 activity may provide a safeguard against oncogenic deregulation of beta-catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression. PMID:10357817

  12. Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in beta-catenin that are independent of beta-catenin mutation status.

    PubMed

    Wang, Rong; Dashwood, W Mohaiza; Bailey, George S; Williams, David E; Dashwood, Roderick H

    2006-10-10

    Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in beta-catenin, but the spectrum of such mutations can be influenced by phytochemicals such as chlorophyllin (CHL) and indole-3-carbinol (I3C). In the present study, we determined the mutation status of beta-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of beta-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in beta-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and beta-catenin degradation. These tumors were found to express a 10-fold range of beta-catenin mRNA levels, independent of the beta-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, beta-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of beta-catenin/Tcf signaling. Tumors with the highest levels of beta-catenin mRNA often had over-expressed beta-catenin protein, and those with lower beta-catenin mRNA typically had low beta-catenin protein expression, but there were exceptions (high beta-catenin mRNA/low beta-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize beta-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of beta-catenin itself at the mRNA level, contributing to high beta-catenin protein levels. Similar findings have been reported in primary human colon cancers and their liver metastases, compared with matched normal-looking tissue. Thus, further studies are warranted on the mechanisms that upregulate beta-catenin at the transcriptional level in human and rodent colon cancers.

  13. Multiple roles of mesenchymal beta-catenin during murine limb patterning.

    PubMed

    Hill, Theo P; Taketo, Makoto M; Birchmeier, Walter; Hartmann, Christine

    2006-04-01

    Recently canonical Wnt signaling in the ectoderm has been shown to be required for maintenance of the apical ectodermal ridge (AER) and for dorsoventral signaling. Using conditional gain- and loss-of-function beta-catenin alleles, we have studied the role of mesenchymal beta-catenin activity during limb development. Here, we show that loss of beta-catenin results in limb truncations due to a defect in AER maintenance. Stabilization of beta-catenin also results in truncated limbs, caused by a premature regression of the AER. Concomitantly, in these limbs, the expression of Bmp2, Bmp4 and Bmp7, and of the Bmp target genes Msx1, Msx2 and gremlin, is expanded in the mesenchyme. Furthermore, we found that the expression of Lmx1b, a gene exclusively expressed in the dorsal limb mesenchyme and involved in dorsoventral patterning, is reduced upon loss of beta-catenin activity and is expanded ventrally in gain-of-function limbs. However, the known ectodermal regulators Wnt7a and engrailed 1 are expressed normally. This suggests that Lmx1b is also regulated, in part, by a beta-catenin-mediated Wnt signal, independent of the non-canoncial Wnt7a signaling pathway. In addition, loss of beta-catenin results in a severe agenesis of the scapula. Concurrently, the expression of two genes, Pax1 and Emx2, which have been implicated in scapula development, is lost in beta-catenin loss-of-function limbs; however, only Emx2 is upregulated in gain-of-function limbs. Mesenchymal beta-catenin activity is therefore required for AER maintenance, and for normal expression of Lmx1b and Emx2.

  14. Parkin protects dopaminergic neurons from excessive Wnt/beta-catenin signaling.

    PubMed

    Rawal, Nina; Corti, Olga; Sacchetti, Paola; Ardilla-Osorio, Hector; Sehat, Bita; Brice, Alexis; Arenas, Ernest

    2009-10-23

    Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates beta-catenin protein levels in vivo. Stabilization of beta-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neurons in parkin null animals, suggesting that both increased stabilization and decreased degradation of beta-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and beta-catenin-induced cell death.

  15. Parkin protects dopaminergic neurons from excessive Wnt/{beta}-catenin signaling

    SciTech Connect

    Rawal, Nina; Corti, Olga; Sacchetti, Paola; Ardilla-Osorio, Hector; Sehat, Bita; Brice, Alexis; Arenas, Ernest

    2009-10-23

    Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates {beta}-catenin protein levels in vivo. Stabilization of {beta}-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neurons in parkin null animals, suggesting that both increased stabilization and decreased degradation of {beta}-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and {beta}-catenin-induced cell death.

  16. [Dual-role regulations of canonical Wnt/beta-catenin signaling pathway].

    PubMed

    Liu, Yang; Zhang, Chen-guang; Zhou, Chun-yan

    2010-04-18

    In recent years, Wnt/beta-catenin signaling has been identified as a key player in embryogenesis and human diseases. Canonical Wnt signaling pathway is controlled by a variety of classic molecules like Wnt, beta-catenin, Axin, APC, GSK-3beta and CK1, which interact and coordinate to regulate the expressions of cell signaling molecules. The latest evidences suggest that some components of the Wnt/beta-catenin signaling, like APC, GSK-3beta, CK1, Dkk2 and WISE, play dual roles different from what they have been thought previously. Here we reviewed some recent discoveries on the canonical Wnt/beta-catenin signaling pathway to provide some new ideas and principles for signaling transduction studies.

  17. Beta-Catenin Signaling in Hepatic Development and Progenitors: Which Way Does the WNT Blow?

    PubMed Central

    Lade, Abigale G.; Monga, Satdarshan P. S.

    2015-01-01

    The Wnt/β-catenin pathway is an evolutionarily conserved signaling cascade that plays key roles in development and adult tissue homeostasis and is aberrantly activated in many tumors. Over a decade of work in mouse, chick, xenopus, and zebrafish models has uncovered multiple functions of this pathway in hepatic pathophysiology. Specifically, beta-catenin, the central component of the canonical Wnt pathway, is implicated in the regulation of liver regeneration, development, and carcinogenesis. Wnt-independent activation of beta-catenin by receptor tyrosine kinases has also been observed in the liver. In liver development across various species, through regulation of cell proliferation, differentiation, and maturation, beta-catenin directs foregut endoderm specification, hepatic specification of the foregut, and hepatic morphogenesis. Its role has also been defined in adult hepatic progenitors or oval cells especially in their expansion and differentiation. Thus, beta-catenin undergoes tight temporal regulation to exhibit pleiotropic effects during hepatic development and in hepatic progenitor biology. PMID:21337461

  18. Wnt/beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression

    DTIC Science & Technology

    2014-07-01

    Axis Controls Prostate Cancer Progression PRINCIPAL INVESTIGATOR: Xiuping Yu CONTRACTING ORGANIZATION: Vanderbilt University...COVERED 01 July 2013 - 30 June 2014 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Wnt/beta-Catenin, Foxa2, and CXCR4 axis controls prostate cancer ...development of castration resistant prostate cancer . Our previous studies have shown that Foxa2 is a Wnt/beta-catenin target gene in prostates. Our

  19. Tyrosine residues 654 and 670 in {beta}-cat enin are crucial in regulation of Met-{beta}-catenin interactions

    SciTech Connect

    Zeng, Gang; Apte, Udayan; Micsenyi, Amanda; Bell, Aaron; Monga, Satdarshan P.S. . E-mail: smonga@pitt.edu

    2006-11-01

    {beta}-catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-{beta}-catenin dissociation and nuclear translocation of {beta}-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-{beta}-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in {beta}-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-{beta}-catenin except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of {beta}-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of {beta}-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in {beta}-catenin are crucial in HGF-mediated {beta}-catenin translocation, activation and mitogenesis.

  20. TGF-{beta} modulates {beta}-Catenin stability and signaling in mesenchymal proliferations

    SciTech Connect

    Amini Nik, Saeid; Ebrahim, Rasoul Pour; Dam, Kim van; Cassiman, Jean-Jacques; Tejpar, Sabine . E-mail: sabine.tejpar@med.kuleuven.be

    2007-08-01

    Here for the first time we showed, despite the oncogenic mutations in {beta}-Catenin, that TGF-{beta} is a modulator of {beta}-Catenin levels in tumoral fibroblasts as well as non-tumoral fibroblasts. The results show that the TGF-{beta} pathway is active in desmoids cells and in in situ tumors. A dose dependent increase in {beta}-Catenin protein levels was observed after TGF-{beta} treatment in combination with an increased repression of GSK-3{beta} both in normal and tumoral fibroblasts. TGF-{beta} stimulation also led to an altered - up to 5 fold - transcriptional activity of {beta}-Catenin responsive promoters, such as IGFBP6 as well as increase of TOPflash activity. TGF-{beta} stimulation increased cell proliferation and BrdU incorporation 2.5 times. Taken together, we propose that TGF-{beta} is a modulator of {beta}-Catenin levels in tumoral fibroblasts and non-tumoral fibroblasts, despite the oncogenic mutations already present in this gene in tumoral fibroblasts of desmoid tumors. This modulation of {beta}-Catenin levels by TGF-{beta} may be involved in determining the tumoral phenotype of the cells.

  1. Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium.

    PubMed

    Schneider, Fabian T; Schänzer, Anne; Czupalla, Cathrin J; Thom, Sonja; Engels, Knut; Schmidt, Mirko H H; Plate, Karl H; Liebner, Stefan

    2010-07-01

    Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

  2. Evaluation of myosin VI, E-cadherin and beta-catenin immunostaining in renal cell carcinoma

    PubMed Central

    2010-01-01

    Background Renal cell carcinoma (RCC) is a cancer of increasing incidence and mortality. Currently, there are no immunohistochemical prognostic markers for RCCs in routine use. The aim of this study was to examine for the first time the immunostaining of myosin VI in RCCs as well as its association with E-cadherin and beta-catenin immunostaining and the prognostic significance of these markers in RCCs. Methods Our study population consisted of 152 patients who underwent surgery for RCCs between 1990 and 1999. The tumours were examined with three immunohistochemical markers: myosin VI, E-cadherin and beta-catenin. Results The immunostaining for cytoplasmic myosin VI was common (72%). One-third of the tumours were immunopositive for nuclear myosin VI. Cytoplasmic myosin VI immunopositivity and nuclear beta-catenin immunostaining were associated with lower Fuhrman grades (p = 0.04 and p = 0.005, respectively), but not stages. There was no significant association between myosin VI immunostaining and the histological subtype of RCC. Nuclear myosin VI was associated with the nuclear expression of beta-catenin. A direct association could also be proven between membranous E-cadherin and cytoplasmic beta-catenin. Cytoplasmic myosin VI immunostaining was a marker of poorer prognosis in multivariate Cox regression model adjusted with stage and Fuhrman grade with hazard ratio 2.4 (95% confidence interval 1.1 to 5.0 with p = 0.024). Conclusions Cytoplasmic myosin VI immunopositivity and nuclear beta-catenin immunostaining were associated with lower Fuhrman grades, and there was a strong positive relationship between E-cadherin immunostaining and beta-catenin immunostaining in RCCs. Cytoplasmic myosin VI immunostaining was associated with poorer prognosis in RCCs. PMID:20074327

  3. Nuclear accumulation of beta-catenin occurs commonly in the epithelial cells of juvenile polyps.

    PubMed

    Iwamoto, Michiko; Hoffenberg, Edward J; Carethers, John M; Doctolero, Ryan; Tajima, Akihiro; Sugano, Kentaro; Franklin, Wilbur A; Ahnen, Dennis J

    2005-01-01

    In the two conditions juvenile polyps (JPs) and juvenile polyposis coli (JPC), colonic polyps may have overlapping histologic and phenotypic appearance, but JPC confers a significant risk for colon adenocarcinoma. Although not thought to contain adenomatous polyposis coli (APC) mutations, the status of beta-catenin and full-length APC protein expression in JPs is not known. We evaluated beta-catenin and full-length APC protein expression in JPs from children with JPs and JPC. Cases were identified through endoscopic procedure records. Immunohistochemistry was performed for beta-catenin and full-length APC protein. Loss of heterozygosity at the APC gene locus on chromosome 5 was assessed using two APC-linked microsatellite markers. Polyp and normal colonic tissue were analyzed from 36 children with JPs and 9 with JPC. Both APC and beta-catenin immunoreactivity were present in epithelial cells from all samples but in different patterns. In all normal colon and polyp samples, APC expression was cytoplasmic with maximal immunoreactivity in the goblet cells. In contrast, beta-catenin immunoreactivity in epithelial cells was limited to the plasma membrane in normal colon but was both cytoplasmic and nuclear in all 45 JPs. No evidence of APC gene loss of heterozygosity was found. In polyps from children with JPs and JPC, nuclear beta-catenin accumulation is a consistent feature, and it is not due to APC gene mutation or loss of full-length APC protein expression. Thus, beta-catenin accumulation may be intrinsic to the formation of juvenile-type polyps through an as-yet-undefined mechanism.

  4. Beta-catenin up-regulates the expression of the urokinase plasminogen activator in human colorectal tumors.

    PubMed

    Hiendlmeyer, Elke; Regus, Susanne; Wassermann, Stella; Hlubek, Falk; Haynl, Angela; Dimmler, Arno; Koch, Claudia; Knoll, Claudia; van Beest, Moniek; Reuning, Ute; Brabletz, Thomas; Kirchner, Thomas; Jung, Andreas

    2004-02-15

    Expression of the urokinase plasminogen activator (uPA) increases during the progression of colorectal tumors from adenomas to carcinomas. The highest amounts of uPA are found at the invasion front of carcinomas, which also displays a strong expression of nuclear beta-catenin and is therefore a region expressing beta-catenin target genes at high levels. Here we show that beta-catenin contributes to the transactivation of uPA. Therefore, beta-catenin might have an impact on the capacity of colorectal tumors for invasion and metastasis, as well as dormancy, which are hallmarks of cancer.

  5. MAPK/ERK and Wnt/{beta}-Catenin pathways are synergistically involved in proliferation of Sca-1 positive hepatic progenitor cells

    SciTech Connect

    Jin, Caixia; Samuelson, Lisa; Cui, Cai-Bin; Sun, Yangzhong; Gerber, David A.

    2011-06-17

    Highlights: {yields} Activation of MAPK/ERK pathway with epidermal growth factor (EGF) significantly increased Sca-1{sup +} HPC proliferation and colony formation. {yields} Activation of either IL-6/STAT3 or Wnt/{beta}-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. {yields} Wnt/{beta}-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation and maintain long-term HPCs in vitro. -- Abstract: Hepatic progenitor cells (HPCs) persist in adulthood and have the potential to play a major role in regenerating diseased liver. However, the signaling pathways that both directly and indirectly regulate HPCs' self-renewal and differentiation remain elusive. Previously, we identified a bipotent, stem cell antigen-1 (Sca-1) positive HPC population from naive adult liver tissue. In the present study, we aimed to investigate the involvement of various signaling pathways in Sca-1{sup +} HPC proliferation. Epidermal growth factor (EGF) supplementation shows a significant increase in Sca-1{sup +} HPC proliferation and colony formation while stimulating phosphorylation of ERK1/2 and activating the induction of Cyclin D1. There were no demonstrable effects of EGF on Akt. The MEK inhibitor, PD0325901, inhibits proliferation and ERK1/2 phosphorylation while also suppressing the expression of Cyclin D1. In addition, activation of either IL-6/STAT3 or Wnt/{beta}-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. The Wnt/{beta}-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation ratio and maintain long-term HPC in vitro. The data indicates that the MAPK/ERK pathway is both essential and critical for HPC proliferation, and the Wnt signaling pathway is not sufficient, while it works synergistically with the MAPK/ERK signaling pathway to promote HPC proliferation.

  6. Lack of Muc1-regulated beta-catenin stability results in aberrant expansion of CD11b+Gr1+ myeloid derived suppressor cells from the bone marrow

    PubMed Central

    Poh, Tze Wei; Bradley, Judy M.; Mukherjee, Pinku; Gendler, Sandra J.

    2009-01-01

    Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous population of myeloid cells that inhibit T cell activity and contribute to the immune suppression characteristic of most tumors. We discovered that bone marrow (BM) progenitor cells from the Muc1 knockout (KO) mice differentiated into CD11b+Gr1+ MDSCs in vitro under GM-CSF and IL-4 signaling. MUC1 is a tumor-associated mucin and its cytoplasmic tail (MUC1-CT) can regulate beta-catenin to promote oncogenesis. Given the importance of beta-catenin in hematopoiesis, we hypothesized that the MUC1 regulation of beta-catenin is important for MDSC development. Our current study shows that the aberrant development of BM progenitors into CD11b+Gr1+ MDSCs is dependent on the down regulation of beta-catenin levels that occurs in the absence of Muc1. In light of this, KO mice showed enhanced EL4 tumor growth and were able to better tolerate allogeneic BM185 tumor growth, with an accumulation of CD11b+Gr1+ cells in the blood and tumor draining lymph nodes. WT mice were able to similarly tolerate allogeneic tumor growth when they were injected with CD11b+Gr1+ cells from tumor-bearing KO mice, suggesting that tolerance of allogeneic tumors is dependent on MDSC-mediated immune suppression. This further delineates the ability of Muc1 to control MDSC development which could directly impact tumorigenesis. Knowledge of the biology by which Muc1 regulates the development of myeloid progenitors into MDSCs would also be very useful in enhancing the efficacy of cancer vaccines in the face of tumor immune suppression. PMID:19351842

  7. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  8. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    2001-10-09

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  9. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    1999-01-01

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  10. beta-Catenin in early development of the lancelet embryo indicates specific determination of embryonic polarity.

    PubMed

    Yasui, Kinya; Li, Guorong; Wang, Yong; Saiga, Hidetoshi; Zhang, Peijun; Aizawa, Shinichi

    2002-12-01

    The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

  11. Activation of the canonical Wnt/{beta}-catenin pathway enhances monocyte adhesion to endothelial cells

    SciTech Connect

    Lee, Dong Kun . E-mail: leedk@memorialhealthsource.com; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-08-18

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/{beta}-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3{beta} or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/{beta}-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/{beta}-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules.

  12. An ancient role for nuclear beta-catenin in the evolution of axial polarity and germ layer segregation

    NASA Technical Reports Server (NTRS)

    Wikramanayake, Athula H.; Hong, Melanie; Lee, Patricia N.; Pang, Kevin; Byrum, Christine A.; Bince, Joanna M.; Xu, Ronghui; Martindale, Mark Q.

    2003-01-01

    The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

  13. Beta-catenin/TCF Pathway and Castrate Resistant Progression in Osteoblastic Bone Metastases

    DTIC Science & Technology

    2010-06-01

    beta-catenin complex (1)); fibroblast growth factor 19 ( FGF19 , a high-affinity ligand for FGFR4 (2)); NOTUM, WNT11, WISP1 (regulator [NOTUM (3)] and...we initially selected FGF19 , NOTUM, WNT11, HA synthase 3, and HA synthase 2 and assessed their expression in MDA PCa 118 cells transfected with beta...regulation of cells or are components of the ECM (Table 1). We confirmed that both beta-catenin and FGF19 were reduced by about 3.8 times. Table 1. Gene

  14. Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis.

    PubMed

    Patil, Mohini A; Lee, Susie A; Macias, Everardo; Lam, Ernest T; Xu, Chuanrui; Jones, Kirk D; Ho, Coral; Rodriguez-Puebla, Marcelo; Chen, Xin

    2009-01-01

    Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

  15. Regulation of AR and (beta)-Catenin Signaling by Pin 1 in Prostate Cancer

    DTIC Science & Technology

    2006-10-01

    phosphatidylinositol 3 - kinase AKT pathway in human cancer . Nat. Rev. Cancer 2:489–501. 51. Voeller, H. J., C. I. Truica, and E. P. Gelmann. 1998. Beta-catenin...4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111:241–250. 50. Vivanco, I., and C. L. Sawyers. 2002. The

  16. Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    Transdisciplinary Research in Epigenetics and Cancer Journal Clubs and Transdisciplinary Science Meetings, biweekly and monthly 3. To gain expertise...Target Genes in Prostate and Prostate Cancer PRINCIPAL INVESTIGATOR: Laura Lamb CONTRACTING ORGANIZATION: Washington University...TITLE AND SUBTITLE Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer 5a. CONTRACT NUMBER Genes in

  17. Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation.

    PubMed

    Palma, Icela; Peña, Rocio-Yolanda; Contreras, Alejandra; Ceballos-Reyes, Guillermo; Coyote, Ninel; Eraña, Luis; Kofman-Alfaro, Susana; Queipo, Gloria

    2008-05-18

    Gonadoblastoma (GB) is an in situ tumor consisting of a heterogeneous population of mature and immature germ cells, other cells resembling immature Sertoli/granulosa cells, and Leydig/lutein-like cells, may also be present. GB almost exclusively affects a subset of patients with intersex disorders and in 30% of them overgrowth of the germinal component of the tumor is observed and the lesion is term dysgerminoma/seminoma. Several pathways have been proposed to explain the malignant process, and abnormal OCT3/4 expression is the most robust risk factor for malignant transformation. Some authors have suggested that OCT3/4 and beta-catenin might both be involved in the same oncogenic pathway, as both genes are master regulators of cell differentiation and, overexpression of either gene may result in cancer development. The mechanism by which beta-catenin participates in GB transformation is not completely clear and exploration of the E-cadherin pathway did not conclusively show that this pathway participated in the molecular pathogenesis of GB. Here we analyze seven patients with mixed gonadal dysgenesis and GB, in an effort to elucidate the participation of beta-catenin and E-cadherin, as well as OCT3/4, in the oncogenic pathways involved in the transformation of GB into seminoma/dysgerminoma. We conclude that the proliferation of immature germ cells in GB may be due to an interaction between OCT3/4 and accumulated beta-catenin in the nuclei of the immature germ cells.

  18. Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.

    PubMed Central

    Munemitsu, S; Albert, I; Souza, B; Rubinfeld, B; Polakis, P

    1995-01-01

    The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. We examined the effects of exogenous APC expression on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC caused a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction was due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lacked this activity. Mutational analysis revealed that the central region of the APC protein, which is typically deleted or severely truncated in tumors, was responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7708772

  19. {beta}-Catenin stabilization imparts crypt progenitor phenotype to hyperproliferating colonic epithelia

    SciTech Connect

    Sellin, Joseph H.; Wang Yu; Singh, Pomila; Umar, Shahid

    2009-01-01

    Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we provide mechanistic basis of changes in {beta}-catenin/APC/CKI{epsilon} leading to progression and/or regression of hyperplasia in vivo. In response to CR-induced TMCH, crypt lengths increased significantly between days 6-27 post-infection, followed by a steep decline by day 34. {beta}-Cat{sup 45}/total {beta}-catenin were elevated on day 1 post-infection, preceding changes in crypt length, and persisted for 27 days before declining by day 34. Importantly, cellular CKI{epsilon} and {beta}-catenin co-immunoprecipitated and exhibited remarkable parallel changes in kinetics during hyperplasia/regression phases. {beta}-catenin, phosphorylated at Ser33,37 and Thr41 ({beta}-cat{sup 33,37/41}), was low till day 12, followed by gradual increase until day 27 before declining by day 34. GSK-3{beta} exhibited significant Ser{sup 9}-phosphorylation/inactivation at days 6-12 with partial recovery at days 27-34. Wild type (wt) APC (p312) levels increased at day 6 with transient proteolysis/truncation to p130 form between days 12 and 15; p312 reappeared by day 19 and returned to baseline by day 34. The kinetics of {beta}-Cat{sup 45}/{beta}-catenin nuclear accumulation and acetylation (Ac-{beta}-Cat{sup Lys49}) from days 6 to 27, followed by loss of phosphorylation/acetylation by day 34 was almost identical; Tcf-4 co-immunoprecipitated with {beta}-Cat{sup 45}/{beta}-catenin and localized immunohistochemically to {beta}-Cat{sup 41/45}-positive regions leading to elevated cyclin D1 expression, during the hyperproliferative, but not regression phases of TMCH. CKI{epsilon} mediated phosphorylation of {beta}-Cat{sup 45}, resulting in stabilization/nuclear translocation of {beta}-Cat{sup 45} may be critical for maintaining proliferation at days 6-27. Reversal of GSK-3{beta} phosphorylation and APC changes may be equally critical during the regression phase from days 27 to 34.

  20. [CELL CONTACT PROTEIN BETA-CATENIN IN EPENDYMAL AND EPITHELIAL CELLS OF THE CHOROID PLEXUS OF THE CEREBRAL LATERAL VENTRICLES].

    PubMed

    Kirik, O V; Sufieyva, D A; Nazarenkova, A V; Korzhevskiy, D E

    2016-01-01

    The purpose of this study was to examine the distribution pattern of cellular contacts protein beta-catenin in the choroid plexus and ependyma of lateral ventricles of the brain. The study was conducted on frontal sections of the brain of Wistar rats (n = 10) using polyclonal antibodies against beta-catenin. The obtained preparations were analyzed by microscopy in transmitted light and using confocal laser microscopy. To study the distribution of beta-catenin in different projections, three-dimensional reconstruction was performed. The study demonstrated different distribution patterns of this protein in ependyma and choroid plexus. Unlike ependyma, in the cells of the choroid plexus beta-catenin was distributed in the same way as in simple epithelial tissues (on the basal and lateral borders of the cells). This may indicate different tissue attribution of the ependyma and the choroid plexus epithelium, despite their common origin.

  1. Pituitary gland and beta-catenin signaling: from ontogeny to oncogenesis.

    PubMed

    Gueorguiev, Maria; Grossman, Ashley B

    2009-01-01

    Although pituitary tumors are mostly benign, they share certain molecular events with more malignant neoplasia, although their precise pathogenesis is far from established. The acquisition of new functional characteristics during their evolution suggests a multistep process that leads to tumor transformation. Mutations in classical tumor suppressor genes or oncogenes are infrequently associated with pituitary tumorigenesis. However, alterations in different signaling pathways, especially those involved in pituitary gland development, have emerged as significant features in pituitary adenomas. In particular, changes in inhibitory components of the beta-catenin pathway and its relationship to the cadherin family of peptides may well play an important role in tumorigenesis. We review and assess the role of the beta-catenin signaling pathway in the pathogenesis of pituitary adenomas.

  2. The emergence of pattern in embryogenesis: regulation of beta-catenin localization during early sea urchin development.

    PubMed

    Ettensohn, Charles A

    2006-11-14

    The accumulation of beta-catenin in the nuclei of blastomeres at one pole of the early embryo is a highly conserved and essential feature of animal development. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres during early cleavage and activates gene regulatory networks that drive mesoderm and endoderm formation. Measurements of beta-catenin half-life in vivo have demonstrated a gradient in stability along the animal-vegetal axis. Dishevelled (Dsh), a protein that regulates beta-catenin turnover, is localized in the vegetal cortex, where it has an essential role in stabilizing beta-catenin and activating endomesodermal gene networks. Two motifs of Dsh are required for targeting to the vegetal cortex. Overexpression of Dsh in animal blastomeres does not alter their fate, which suggests that a localized activator of Dsh may be missing in these cells. Wnt signaling may be localized in the early sea urchin embryo, as it is in Xenopus, but findings point to possible differences in the initial polarizing signal in amphibians and echinoderms. Further studies will be required to determine the extent to which mechanisms that control beta-catenin nuclearization in early embryogenesis have been conserved during animal evolution.

  3. Wnt/beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression

    DTIC Science & Technology

    2015-07-01

    bone metastasis and in the development of castration resistant prostate cancer. Our previous studies have shown that Foxa2 is a Wnt/beta-catenin...target gene in prostates. Our preliminary study suggests a Wnt−Foxa2−CXCR4 axis that is involved in PCa bone metastasis, and activation of this axis...promotes progression to CRPCa and facilitates bone colonization by PCa cells, and that targeting this axis will provide a novel treatment for PCa bone

  4. MicroRNA-1826 directly targets beta-catenin (CTNNB1) and MEK1 (MAP2K1) in VHL-inactivated renal cancer.

    PubMed

    Hirata, Hiroshi; Hinoda, Yuji; Ueno, Koji; Nakajima, Koichi; Ishii, Nobuhisa; Dahiya, Rajvir

    2012-03-01

    The aim of this project is to identify new therapeutic microRNAs (miRNAs) for von Hippel-Lindau (VHL)-inactivated renal cancer cells. We initially identified several potential miRNAs targeting CTNNB1 and MEK1 using several targets scan algorithms. Only miR-1826 was found to target CTNNB1 and MEK1. Therefore, we focused on miRNA-1826 and performed 3' untranslated region (UTR) luciferase assay, functional analyses and association study between miR-1826 expression and renal cancer patient outcomes. miR-1826 expression was significantly lower in renal cancer tissues compared with non-neoplastic areas and lower expression was significantly associated with overall shorter survival and earlier recurrence after radical nephrectomy. Following miR-1826 transfection, 3' UTR luciferase activity and protein expression of beta-catenin and MEK1 were significantly downregulated in renal cancer cells. Introduction of miR-1826 also inhibited renal cancer cell proliferation, invasion and migration. Additionally, miR-1826 promoted apoptosis and G(1) arrest in VHL-inactivated renal cancer cells. Knockdowns of CTNNB1 and MEK1 by small interfering RNAs reproduced the tumor-suppressive effect of miR-1826. Our data suggest that the miR-1826 plays an important role as a tumor suppressor by downregulating beta-catenin and MEK1 in VHL-inactivated renal cancers.

  5. MicroRNA-1826 directly targets beta-catenin (CTNNB1) and MEK1 (MAP2K1) in VHL-inactivated renal cancer

    PubMed Central

    Hirata, Hiroshi; Hinoda, Yuji; Ueno, Koji; Nakajima, Koichi; Ishii, Nobuhisa; Dahiya, Rajvir

    2012-01-01

    The aim of this project is to identify new therapeutic microRNAs (miRNAs) for von Hippel-Lindau (VHL)-inactivated renal cancer cells. We initially identified several potential miRNAs targeting CTNNB1 and MEK1 using several targets scan algorithms. Only miR-1826 was found to target CTNNB1 and MEK1. Therefore, we focused on miRNA-1826 and performed 3′ untranslated region (UTR) luciferase assay, functional analyses and association study between miR-1826 expression and renal cancer patient outcomes. miR-1826 expression was significantly lower in renal cancer tissues compared with non-neoplastic areas and lower expression was significantly associated with overall shorter survival and earlier recurrence after radical nephrectomy. Following miR-1826 transfection, 3′ UTR luciferase activity and protein expression of beta-catenin and MEK1 were significantly downregulated in renal cancer cells. Introduction of miR-1826 also inhibited renal cancer cell proliferation, invasion and migration. Additionally, miR-1826 promoted apoptosis and G1 arrest in VHL-inactivated renal cancer cells. Knockdowns of CTNNB1 and MEK1 by small interfering RNAs reproduced the tumor-suppressive effect of miR-1826. Our data suggest that the miR-1826 plays an important role as a tumor suppressor by downregulating beta-catenin and MEK1 in VHL-inactivated renal cancers. PMID:22180573

  6. Loss of p53 expression is accompanied by upregulation of beta-catenin in meningiomas: a concomitant reciprocal expression.

    PubMed

    Pećina-Šlaus, Nives; Kafka, Anja; Vladušić, Tomislav; Tomas, Davor; Logara, Monika; Skoko, Josip; Hrašćan, Reno

    2016-04-01

    Crosstalk between Wnt and p53 signalling pathways in cancer has long been suggested. Therefore in this study we have investigated the involvement of these pathways in meningiomas by analysing their main effector molecules, beta-catenin and p53. Cellular expression of p53 and beta-catenin proteins and genetic changes in TP53 were analysed by immunohistochemistry, PCR/RFLP and direct sequencing of TP53 exon 4. All the findings were analysed statistically. Our analysis showed that 47.5% of the 59 meningiomas demonstrated loss of expression of p53 protein. Moderate and strong p53 expression in the nuclei was observed in 8.5% and 6.8% of meningiomas respectively. Gross deletion of TP53 gene was observed in one meningioma, but nucleotide alterations were observed in 35.7% of meningiomas. In contrast, beta-catenin, the main Wnt signalling molecule, was upregulated in 71.2%, while strong expression was observed in 28.8% of meningiomas. The concomitant expressions of p53 and beta-catenin were investigated in the same patients. In the analysed meningiomas, the levels of the two proteins were significantly negatively correlated (P = 0.002). This indicates that meningiomas with lost p53 upregulate beta-catenin and activate Wnt signalling. Besides showing the reciprocal relationship between proteins, we also showed that the expression of p53 was significantly (P = 0.021) associated with higher meningioma grades (II and III), while beta-catenin upregulation was not associated with malignancy grades. Additionally, women exhibited significantly higher values of p53 loss when compared to males (P = 0.005). Our findings provide novel information about p53 involvement in meningeal brain tumours and reveal the complex relationship between Wnt and p53 signalling, they suggest an important role for beta-catenin in these tumours.

  7. Beta-catenin deficiency causes DiGeorge syndrome-like phenotypes through regulation of Tbx1.

    PubMed

    Huh, Sung-Ho; Ornitz, David M

    2010-04-01

    DiGeorge syndrome (DGS) is a common genetic disease characterized by pharyngeal apparatus malformations and defects in cardiovascular, craniofacial and glandular development. TBX1 is the most likely candidate disease-causing gene and is located within a 22q11.2 chromosomal deletion that is associated with most cases of DGS. Here, we show that canonical Wnt-beta-catenin signaling negatively regulates Tbx1 expression and that mesenchymal inactivation of beta-catenin (Ctnnb1) in mice caused abnormalities within the DGS phenotypic spectrum, including great vessel malformations, hypoplastic pulmonary and aortic arch arteries, cardiac malformations, micrognathia, thymus hypoplasia and mislocalization of the parathyroid gland. In a heterozygous Fgf8 or Tbx1 genetic background, ectopic activation of Wnt-beta-catenin signaling caused an increased incidence and severity of DGS-like phenotypes. Additionally, reducing the gene dosage of Fgf8 rescued pharyngeal arch artery defects caused by loss of Ctnnb1. These findings identify Wnt-beta-catenin signaling as a crucial upstream regulator of a Tbx1-Fgf8 signaling pathway and suggest that factors that affect Wnt-beta-catenin signaling could modify the incidence and severity of DGS.

  8. Calcium/calmodulin-dependent protein kinase type IV is a target gene of the Wnt/beta-catenin signaling pathway.

    PubMed

    Arrázola, Macarena S; Varela-Nallar, Lorena; Colombres, Marcela; Toledo, Enrique M; Cruzat, Fernando; Pavez, Leonardo; Assar, Rodrigo; Aravena, Andrés; González, Mauricio; Montecino, Martín; Maass, Alejandro; Martínez, Servet; Inestrosa, Nibaldo C

    2009-12-01

    Calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. The expression of CaMKIV and the activation of CREB regulated genes are involved in memory and neuronal survival. We report here that: (a) a bioinformatic analysis of 15,476 promoters of the human genome predicted several Wnt target genes, being CaMKIV a very interesting candidate; (b) CaMKIV promoter contains TCF/LEF transcription motifs similar to those present in Wnt target genes; (c) biochemical studies indicate that lithium and the canonical ligand Wnt-3a induce CaMKIV mRNA and protein expression levels in rat hippocampal neurons as well as CaMKIV promoter activity; (d) treatment of hippocampal neurons with Wnt-3a increases the binding of beta-catenin to the CaMKIV promoter: (e) In vivo activation of the Wnt signaling improve spatial memory impairment and restores the expression of CaMKIV in a mice double transgenic model for Alzheimer's disease which shows decreased levels of the kinase. We conclude that CaMKIV is regulated by the Wnt signaling pathway and that its expression could play a role in the neuroprotective function of the Wnt signaling against the Alzheimer's amyloid peptide.

  9. Pleiotrophin regulates lung epithelial cell proliferation and differentiation during fetal lung development via beta-catenin and Dlk1.

    PubMed

    Weng, Tingting; Gao, Li; Bhaskaran, Manoj; Guo, Yujie; Gou, Deming; Narayanaperumal, Jeyaparthasarathy; Chintagari, Narendranath Reddy; Zhang, Kexiong; Liu, Lin

    2009-10-09

    The role of pleiotrophin in fetal lung development was investigated. We found that pleiotrophin and its receptor, protein-tyrosine phosphatase receptor beta/zeta, were highly expressed in mesenchymal and epithelial cells of the fetal lungs, respectively. Using isolated fetal alveolar epithelial type II cells, we demonstrated that pleiotrophin promoted fetal type II cell proliferation and arrested type II cell trans-differentiation into alveolar epithelial type I cells. Pleiotrophin also increased wound healing of injured type II cell monolayer. Knockdown of pleiotrophin influenced lung branching morphogenesis in a fetal lung organ culture model. Pleiotrophin increased the tyrosine phosphorylation of beta-catenin, promoted beta-catenin translocation into the nucleus, and activated T cell factor/lymphoid enhancer factor transcription factors. Dlk1, a membrane ligand that initiates the Notch signaling pathway, was identified as a downstream target of the pleiotrophin/beta-catenin pathway by endogenous dlk1 expression, promoter assay, and chromatin immunoprecipitation. These results provide evidence that pleiotrophin regulates fetal type II cell proliferation and differentiation via integration of multiple signaling pathways including pleiotrophin, beta-catenin, and Notch pathways.

  10. Wnt/beta-catenin signaling plays an essential role in activation of odontogenic mesenchyme during early tooth development.

    PubMed

    Chen, Jianquan; Lan, Yu; Baek, Jin-A; Gao, Yang; Jiang, Rulang

    2009-10-01

    Classical tissue recombination studies demonstrated that initiation of tooth development depends on activation of odontogenic potential in the mesenchyme by signals from the presumptive dental epithelium. Although several members of the Wnt family of signaling molecules are expressed in the presumptive dental epithelium at the beginning of tooth initiation, whether Wnt signaling is directly involved in the activation of the odontogenic mesenchyme has not been characterized. In this report, we show that tissue-specific inactivation of beta-catenin, a central component of the canonical Wnt signaling pathway, in the developing tooth mesenchyme caused tooth developmental arrest at the bud stage in mice. We show that mesenchymal beta-catenin function is required for expression of Lef1 and Fgf3 in the developing tooth mesenchyme and for induction of primary enamel knot in the developing tooth epithelium. Expression of Msx1 and Pax9, two essential tooth mesenchyme transcription factors downstream of Bmp and Fgf signaling, respectively, were not altered in the absence of beta-catenin in the tooth mesenchyme. Moreover, we found that constitutive stabilization of beta-catenin in the developing palatal mesenchyme induced aberrant palatal epithelial invaginations that resembled early tooth buds both morphologically and in epithelial molecular marker expression, but without activating expression of Msx1 and Pax9 in the mesenchyme. Together, these results indicate that activation of the mesenchymal odontogenic program during early tooth development requires concerted actions of Bmp, Fgf and Wnt signaling from the presumptive dental epithelium to the mesenchyme.

  11. MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer

    PubMed Central

    Hirata, Hiroshi; Hinoda, Yuji; Ueno, Koji; Shahryari, Varahram; Tabatabai, Z.Laura; Dahiya, Rajvir

    2012-01-01

    The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer (BC) progression. Tumor-suppressive microRNAs (miRNAs) targeting these cancer pathways may provide a new therapeutic approach for BC. We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC and MEK1 using several target scan algorithms. Also 3′ untranslated region luciferase activity and protein expression of these target genes were significantly downregulated in miR-1826-transfected BC cells (J82 and T24). The expression of miR-1826 was lower in BC tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. Also the expression of miR-1826 was much lower in three BC cell lines (J82, T24 and TCCSUP) compared with a normal bladder cell line (SV-HUC-1). We then performed analyses to look at miR-1826 function and found that miR-1826 inhibited BC cell viability, invasion and migration. We also found increased apoptosis and G1 cell cycle arrest in miR-1826-transfected BC cells. To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knockdown, we knocked down CTNNB1/MEK1 messenger RNA using a small interfering RNA (siRNA) technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in BC cells. In conclusion, our data suggest that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC downregulation in BC. PMID:22049531

  12. MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer.

    PubMed

    Hirata, Hiroshi; Hinoda, Yuji; Ueno, Koji; Shahryari, Varahram; Tabatabai, Z Laura; Dahiya, Rajvir

    2012-01-01

    The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer (BC) progression. Tumor-suppressive microRNAs (miRNAs) targeting these cancer pathways may provide a new therapeutic approach for BC. We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC and MEK1 using several target scan algorithms. Also 3' untranslated region luciferase activity and protein expression of these target genes were significantly downregulated in miR-1826-transfected BC cells (J82 and T24). The expression of miR-1826 was lower in BC tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. Also the expression of miR-1826 was much lower in three BC cell lines (J82, T24 and TCCSUP) compared with a normal bladder cell line (SV-HUC-1). We then performed analyses to look at miR-1826 function and found that miR-1826 inhibited BC cell viability, invasion and migration. We also found increased apoptosis and G(1) cell cycle arrest in miR-1826-transfected BC cells. To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knockdown, we knocked down CTNNB1/MEK1 messenger RNA using a small interfering RNA (siRNA) technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in BC cells. In conclusion, our data suggest that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC downregulation in BC.

  13. Exploration of the APC/beta-catenin (WNT) pathway and a histologic classification system for pulmonary artery intimal sarcoma. A study of 18 cases.

    PubMed

    Gaumann, A; Bode-Lesniewska, B; Zimmermann, D R; Fanburg-Smith, J C; Kirkpatrick, C J; Hofstädter, F; Woenckhaus, M; Stoehr, R; Obermann, E C; Dietmaier, W; Hartmann, A

    2008-11-01

    APC, a tumor suppressor gene in the Wnt pathway, stabilizes beta-catenin and controls cell growth. Mutation of APC or beta-catenin leads to nuclear accumulation of beta-catenin and transcription of cyclin D1/cyclin A. Pulmonary artery sarcoma (PAS) were studied by morphologic, immunohistochemical, and molecular genetic methods of the Wnt pathway. Eighteen cases were included: mean age 52 years, primary intraluminal location with typical clinical presentation. PAS were classified as epithelioid (n = 4) or malignant fibrous histiocytoma (MFH; spindled/pleomorphic, n = 4), myxofibrosarcoma (n = 8), and one each hemangiopericytoma-like or malignant inflammatory myofibroblastic tumor-like. The tumor cells demonstrated vimentin, focal actins, and rare focal desmin positivity. All but one were grade 2 or 3 by FNCLCC grading. Alteration in chromosome 5q21 (APC) was found in 4/14 PAS by LOH, mostly epithelioid-type; an MFH-type case demonstrated microsatellite instability (MSI) and nuclear beta-catenin. Cyclin D1 was expressed in seven tumors, all myxofibrosarcoma-type. No mutations were detected in APC or beta-catenin. In summary, PAS are predominantly intermediate grade myxofibrosarcoma in middle-aged males, and fatal in two-thirds of patients. Despite myofibroblastic phenotype, APC/beta-catenin pathway changes are rare. Cyclin D1, only expressed in the myxofibrosarcoma-type, is likely transcribed via factors other than beta-catenin.

  14. Stabilized beta-catenin in thymic epithelial cells blocks thymus development and function.

    PubMed

    Zuklys, Saulius; Gill, Jason; Keller, Marcel P; Hauri-Hohl, Mathias; Zhanybekova, Saule; Balciunaite, Gina; Na, Kyung-Jae; Jeker, Lukas T; Hafen, Katrin; Tsukamoto, Noriyuki; Amagai, Takashi; Taketo, Makoto M; Krenger, Werner; Holländer, Georg A

    2009-03-01

    Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.

  15. Frequent deletions and mutations of the beta-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma.

    PubMed

    Takayasu, H; Horie, H; Hiyama, E; Matsunaga, T; Hayashi, Y; Watanabe, Y; Suita, S; Kaneko, M; Sasaki, F; Hashizume, K; Ozaki, T; Furuuchi, K; Tada, M; Ohnuma, N; Nakagawara, A

    2001-04-01

    Hepatoblastoma (HBL) is the most common malignant liver tumor in young children. Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, we searched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, all of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmic accumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

  16. Beta-catenin Forms Protein Aggregation at High Concentrations in HEK293TCells

    PubMed Central

    Jazi, Marie Saghaeian; Najafi, Seyed Mahmoud Arab

    2017-01-01

    Background: The canonical Wnt signal transduction (or the Wnt/β-catenin pathway) plays a crucial role in the development of animals and in carcinogenesis. Beta-catenin is the central component of this signaling pathway. The activation of Wnt/β-catenin signaling results in the cytoplasmic and nuclear accumulation of β-catenin. In the nucleus, β-catenin interacts with the TCF/LEF transcription factors and, therefore, participates in the upregulation or downregulation of some important genes involved in diverse cellular activities. In addition, β-catenin is a critical component of the cadherin-mediated cell adherens junction. We had previously noticed that very high cellular concentrations of β-catenin had a negative effect on the transcriptional activity of this protein and, therefore, the aim of this study was to find a mechanism for this negative interaction. Methods: Cell fractionation, western blotting, and immunofluorescence microscopy experiments were performed to measure β-catenin protein levels and β-catenin cellular localization in HEK293Tcells transfected with various amounts of a β-catenin-encoding plasmid. Also, total RNA was extracted from the cells and used for reverse transcriptase-PCR experiments to measure the expression of the β-catenin target genes. SPSS, version 16, was used to analyze the results statistically. Results: We demonstrated that overexpression of β-catenin led to the formation of rod-shaped protein aggregates. The aggregate structures were mainly formed in the cell nucleus and were heavy enough to be isolated by centrifugation. Beta-catenin aggregate formation was accompanied by a decrease in the expression of the β-catenin target genes used in this study. Conclusion: Since deregulation of β-catenin function occurs in several human diseases, including cancer and neurological disorders, the results of this paper further support the possible biological and clinical significance of β-catenin aggregate formation. PMID

  17. Negative regulation of the Wnt signal by MM-1 through inhibiting expression of the wnt4 gene.

    PubMed

    Yoshida, Tatsuya; Kitaura, Hirotake; Hagio, Yuko; Sato, Toshiya; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2008-04-01

    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc through TIF1beta/KAP1, a transcriptional corepressor, and that the c-fms gene was a target gene involved in this pathway. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, to further identify target genes of MM-1, DNA microarray analysis was carried out by comparing expression levels of genes in MM-1 knockdown and parental cells, and the wnt4 gene, a member of the Wnt-beta-catenin pathway, was identified as a target gene of MM-1. Increased expression level of the wnt4 gene, accumulation and translocation of beta-catenin to the cytoplasm and nucleus, and upregulation of TCF/Lef-1, a target protein of the Wnt-beta-catenin pathway, were found in MM-1 knockdown cells. Reporter assays using various deletion constructs of the wnt4 gene promoter showed that MM-1 recognized the region spanning -286 to -229 from a transcription start site, and MM-1 complex was found to bind to this region by chromatin immunoprecipitation and gel mobility shift assays. Furthermore, it was found that Egr-1 and MM-1 were bound to this region and that both proteins mutually down-regulate promoter activity of the wnt4 gene. Since the c-myc gene is the target gene of the Wnt-beta-catenin pathway, these findings suggest that MM-1 inhibits c-Myc by a dual mechanism.

  18. Germinated brown rice (GBR) reduces the incidence of aberrant crypt foci with the involvement of beta-catenin and COX-2 in azoxymethane-induced colon cancer in rats.

    PubMed

    Latifah, Saiful Yazan; Armania, Nurdin; Tze, Tan Hern; Azhar, Yaacob; Nordiana, Abdul Hadi; Norazalina, Saad; Hairuszah, Ithnin; Saidi, Moin; Maznah, Ismail

    2010-03-26

    Chemoprevention has become an important area in cancer research due to the failure of current therapeutic modalities. Epidemiological and preclinical studies have demonstrated that nutrition plays a vital role in the etiology of cancer. This study was conducted to determine the chemopreventive effects of germinated brown rice (GBR) in rats induced with colon cancer. GBR is brown rice that has been claimed to be richer in nutrients compared to the common white rice. The male Sprague Dawley rats (6 weeks of age) were randomly divided into 5 groups: (G1) positive control (with colon cancer, unfed with GBR), (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg)), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control (without colon cancer, unfed with GBR). GBR was administered orally once daily via gavage after injection of 15 mg/kg of body weight of azoxymethane (AOM) once a week for two weeks, intraperitonially. After 8 weeks of treatment, animals were sacrificed and colons were removed. Colonic aberrant crypt foci (ACF) were evaluated histopathologically. Total number of ACF and AC, and multicrypt of ACF, and the expression of beta-catenin and COX-2 reduced significantly (p < 0.05) in all the groups treated with GBR (G2, G3 and G4) compared to the control group (G1). Spearman rank correlation test showed significant positive linear relationship between total beta-catenin and COX-2 score (Spearman's rho = 0.616, p = 0.0001). It is demonstrated that GBR inhibits the development of total number of ACF and AC, and multicrypt of ACF, reduces the expression of beta-catenin and COX-2, and thus can be a promising dietary supplement in prevention of colon cancer.

  19. Discovery of Novel Drugs to Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-13-1-0113 TITLE: Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin...Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis 5a...include area code) 1 Table of contents Table of contents 1 Introduction 2 Overall Summary 2 Body 4 β-catenin is activated during tibial

  20. Polyethylenimine-cationized beta-catenin protein transduction activates the Wnt canonical signaling pathway more effectively than cationic lipid-based transduction.

    PubMed

    Kitazoe, Midori; Futami, Junichiro; Nishikawa, Mitsuo; Yamada, Hidenori; Maeda, Yoshitake

    2010-04-01

    The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by beta-catenin protein transduction. Constitutively active beta-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active beta-catenin protein was added to HEK-293 cells, and induction of several Wnt/beta-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active beta-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active beta-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.

  1. Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway.

    PubMed

    Liu, Yu-Ting; Shang, Donghao; Akatsuka, Shinya; Ohara, Hiroki; Dutta, Khokon Kumar; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Izumiya, Masashi; Abe, Kouichiro; Nakagama, Hitoshi; Noguchi, Noriko; Toyokuni, Shinya

    2007-12-01

    Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase zeta or receptor tyrosine phosphatase beta) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16 approximately 552-fold in the RCCs. Network analysis of the expression revealed the involvement of the beta-catenin pathway in the RCCs. In the RCCs, dephosphorylated beta-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear beta-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating beta-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification.

  2. Prenatal cadmium exposure dysregulates sonic hedgehog and Wnt/beta-catenin signaling in the thymus resulting in altered thymocyte development

    SciTech Connect

    Hanson, Miranda L.; Brundage, Kathleen M.; Schafer, Rosana; Tou, Janet C.; Barnett, John B.

    2010-01-15

    Cadmium (Cd) is both an environmental pollutant and a component of cigarette smoke. Although evidence demonstrates that adult exposure to Cd causes changes in the immune system, there are limited reports in the literature of immunomodulatory effects of prenatal exposure to Cd. The sonic hedgehog (Shh) and Wnt/beta-catenin pathways are required for thymocyte maturation. Several studies have demonstrated that Cd exposure affects these pathways in different organ systems. This study was designed to investigate the effect of prenatal Cd exposure on thymocyte development, and to determine if these effects were linked to dysregulation of Shh and Wnt/beta-catenin pathways. Pregnant C57Bl/6 mice were exposed to an environmentally relevant dose (10 ppm) of Cd throughout pregnancy and effects on the thymus were assessed on the day of birth. Thymocyte phenotype was determined by flow cytometry. A Gli:luciferase reporter cell line was used to measure Shh signaling. Transcription of target genes and translation of key components of both signaling pathways were assessed using real-time RT-PCR and western blot, respectively. Prenatal Cd exposure increased the number of CD4{sup +} cells and a subpopulation of double-negative cells (DN; CD4{sup -}CD8{sup -}), DN4 (CD44{sup -}CD25{sup -}). Shh and Wnt/beta-catenin signaling were both decreased in the thymus. Target genes of Shh (Patched1 and Gli1) and Wnt/beta-catenin (c-fos, and c-myc) were affected differentially among thymocyte subpopulations. These findings suggest that prenatal exposure to Cd dysregulates two signaling pathways in the thymus, resulting in altered thymocyte development.

  3. Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a beta-catenin dependent pathway.

    PubMed

    Pillai, Murali C; Vines, Carol A; Wikramanayake, Athula H; Cherr, Gary N

    2003-04-15

    Sea urchin (Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of beta-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC(50) of creosote for complete exogastrulation being 1.57 ppm, while the EC(50)s of the PAHs ranged from 0.41 ppm (2.0 microM) to 4.33 ppm (33.5 microM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against beta-catenin, a multi-functional protein known to be involved in cell-cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of beta-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since beta-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types.

  4. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations.

    PubMed

    Yamamoto, Yoshiya; Sakamoto, Michiie; Fujii, Gen; Tsuiji, Hitomi; Kenetaka, Kengo; Asaka, Masahiro; Hirohashi, Setsuo

    2003-03-01

    To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in beta-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant beta-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with beta-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.

  5. Wnt/{beta}-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    SciTech Connect

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.

  6. Smed-Evi/Wntless is required for beta-catenin-dependent and -independent processes during planarian regeneration.

    PubMed

    Adell, Teresa; Salò, Emili; Boutros, Michael; Bartscherer, Kerstin

    2009-03-01

    Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-beta-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-beta-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through beta-catenin-dependent and -independent pathways.

  7. E-cadherin and beta-catenin expression in breast medullary carcinomas.

    PubMed

    Charpin, C; Bonnier, P; Garcia, S; Andrac, L; Crebassa, B; Dorel, M; Lavaut, M N; Allasia, C

    1999-08-01

    The initial step of cancer invasion and metastasis is the escape of tumour cells from the primary site, involving disruption of normal cell-cell adhesion and E-cadherin (E-cad) and beta-catenin (beta-cat) down-regulation, as shown in various types of human malignancies including breast carcinomas. Medullary carcinomas are high grade and poorly differentiated tumours with syncytial typical pattern, and prognosis unexpectedly better than that in high grade breast carcinomas. In a series of 55 breast typical medullary carcinomas diagnosed according to the strict use of Ridolfi et al (Cancer 40: 1365-1385, 1977) criteria, E-cad and beta-cat were investigated using quantitative (SAMBA 2005 system) immunocytochemical assays on frozen sections. Results were compared to that obtained on paraffin sections and in a series (n=55) of grade 3 ductal carcinomas. It was shown that medullary carcinomas significantly (p<0.001) expressed more E-cad and beta-cat than grade 3 ductal carcinomas. E-cad and beta-cat correlated with high expression of P53, of c-erbB, and of Ki-67 antigens, and with lack of hormone receptors antigenic sites (p<0.001). It was concluded that favourable prognosis and syncytial pattern of typical breast medullary carcinomas likely results, at least partly, from a particular expression of cell-cell adhesion molecules, significantly limiting tumour growth and efficiently mastering the tumour cell dissemination, opposing to high proliferative activity (grade 3).

  8. A subset of high-grade pulmonary neuroendocrine carcinomas shows up-regulation of matrix metalloproteinase-7 associated with nuclear beta-catenin immunoreactivity, independent of EGFR and HER-2 gene amplification or expression.

    PubMed

    Pelosi, Giuseppe; Scarpa, Aldo; Veronesi, Giulia; Spaggiari, Lorenzo; Del Curto, Barbara; Moore, Patrick S; Maisonneuve, Patrick; Sonzogni, Angelica; Masullo, Michele; Viale, Giuseppe

    2005-12-01

    Nuclear translocation of beta-catenin has been correlated with epidermal growth factor receptor (EGFR) overexpression/activation in non-small cell lung cancer. Less is known on beta-catenin transactivation in high-grade pulmonary neuroendocrine tumors and on the status of beta-catenin activating EGFR and human epidermal growth factor receptor 2 (HER-2) or beta-catenin target genes cyclin D1 and matrix metalloproteinase-7 (MMP-7). beta-catenin immunoreactivity was evaluated in 51 large-cell neuroendocrine carcinomas (LCNEC) and 45 small-cell lung carcinomas (SCLC). Nineteen cases were assessed for beta-catenin gene exon 3 mutations, expression of MMP-7, and expression/gene amplification of EGFR, HER-2, and cyclin D1. beta-catenin was expressed in all 96 high-grade neuroendocrine tumors, the vast majority (94%) showing >50% immunopositive cells. A disarrayed immunoreactivity, however, was commonly encountered consisting in variably altered membrane-associated patterns of staining along with progressive accumulation of cytoplasmic immunoreactivity. In LCNEC, but not in SCLC, the disarrayed patterns correlated with EGFR and HER-2 protein expression. beta-catenin nuclear accumulation was found in nine tumors, including seven LCNEC and two SCLC, and was always associated with disarrayed immunoreactivity and increased MMP-7, but not cyclin D1 expression. These cases, however, did not show beta-catenin gene mutations or EGFR and HER-2 gene amplification or expression. No association was found between nuclear beta-catenin and any clinicopathological variable including patients' survival. The subcellular compartmentalization of beta-catenin is profoundly altered in high-grade pulmonary neuroendocrine tumors. A minor subset of these tumors shows beta-catenin nuclear accumulation in association with increased expression of MMP-7, but not of cyclin D1, independent of EGFR and HER-2 gene amplification or expression.

  9. Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice1

    PubMed Central

    Nicol, Barbara; Yao, Humphrey H.-C.

    2015-01-01

    Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development. PMID:26108792

  10. Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice.

    PubMed

    Nicol, Barbara; Yao, Humphrey H-C

    2015-08-01

    Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.

  11. Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

    PubMed

    Amary, Maria Fernanda C; Pauwels, Patrick; Meulemans, Els; Roemen, Guido M; Islam, Lily; Idowu, Bernadine; Bousdras, Konstantinos; Diss, Timothy C; O'Donnell, Paul; Flanagan, Adrienne M

    2007-09-01

    Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

  12. Activation of the Wnt/{beta}-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice

    SciTech Connect

    Chen, Yanchun; Guan, Yingjun; Liu, Huancai; Wu, Xin; Yu, Li; Wang, Shanshan; Zhao, Chunyan; Du, Hongmei; Wang, Xin

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Wnt3a and Cyclin D1 were upregulated in the spinal cord of the ALS mice. Black-Right-Pointing-Pointer {beta}-catenin translocated from the cell membrane to the nucleus in the ALS mice. Black-Right-Pointing-Pointer Wnt3a, {beta}-catenin and Cyclin D1 co-localized for astrocytes were all increased. Black-Right-Pointing-Pointer BrdU/Cyclin D1 double-positive cells were increased in the spinal cord of ALS mice. Black-Right-Pointing-Pointer BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. -- Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however, the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, {beta}-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/{beta}-catenin signaling pathway. We determined the expression of Wnt3a, {beta}-catenin, and Cyclin D1 in the adult spinal cord of SOD1{sup G93A} ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, {beta}-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, {beta}-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, {beta}-catenin or Cyclin D1 in mature GFAP{sup +} astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that

  13. Retinoic acid and Wnt/beta-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus.

    PubMed

    Onai, Takayuki; Lin, Hsiu-Chin; Schubert, Michael; Koop, Demian; Osborne, Peter W; Alvarez, Susana; Alvarez, Rosana; Holland, Nicholas D; Holland, Linda Z

    2009-08-15

    A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

  14. Deciphering the function of canonical Wnt signals in development and disease: conditional loss- and gain-of-function mutations of beta-catenin in mice.

    PubMed

    Grigoryan, Tamara; Wend, Peter; Klaus, Alexandra; Birchmeier, Walter

    2008-09-01

    Wnt signaling is one of a handful of powerful signaling pathways that play crucial roles in the animal life by controlling the genetic programs of embryonic development and adult homeostasis. When disrupted, these signaling pathways cause developmental defects, or diseases, among them cancer. The gateway of the canonical Wnt pathway, which contains >100 genes, is an essential molecule called beta-catenin (Armadillo in Drosophila). Conditional loss- and gain-of-function mutations of beta-catenin in mice provided powerful tools for the functional analysis of canonical Wnt signaling in many tissues and organs. Such studies revealed roles of Wnt signaling that were previously not accessible to genetic analysis due to the early embryonic lethality of conventional beta-catenin knockout mice, as well as the redundancy of Wnt ligands, receptors, and transcription factors. Analysis of conditional beta-catenin loss- and gain-of-function mutant mice demonstrated that canonical Wnt signals control progenitor cell expansion and lineage decisions both in the early embryo and in many organs. Canonical Wnt signaling also plays important roles in the maintenance of various embryonic or adult stem cells, and as recent findings demonstrated, in cancer stem cell types. This has opened new opportunities to model numerous human diseases, which have been associated with deregulated Wnt signaling. Our review summarizes what has been learned from genetic studies of the Wnt pathway by the analysis of conditional beta-catenin loss- and gain-of-function mice.

  15. CD133 expression correlates with membrane beta-catenin and E-cadherin loss from human hair follicle placodes during morphogenesis.

    PubMed

    Gay, Denise L; Yang, Chao-Chun; Plikus, Maksim V; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E; Cotsarelis, George

    2015-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

  16. URG11 mediates hypoxia-induced epithelial-to-mesenchymal transition by modulation of E-cadherin and {beta}-catenin

    SciTech Connect

    Du, Rui; Huang, Chen; Bi, Qian; Zhai, Ying; Xia, Lin; Liu, Jie; Sun, Shiren; Fan, Daiming

    2010-01-01

    Upregulated gene 11 (URG11), recently identified as a new HBx-upregulated gene that may activate {beta}-catenin and Wnt signaling, was found to be upregulated in a human tubule cell line under low oxygen. Here, we investigated the potential role of URG11 in hypoxia-induced renal tubular epithelial-to-mesenchymal (EMT). Overexpression of URG11 in a human proximal tubule cell line (HK2) promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin and {alpha}-SMA, while URG11 knockdown by siRNA effectively reversed hypoxia-induced EMT. URG11 promoted the expression of {beta}-catenin and increased its nuclear accumulation under normoxic conditions through transactivation of the {beta}-catenin promoter. This in turn upregulated {beta}-catenin/T-cell factor (TCF) and its downstream effector genes, vimentin, and {alpha}-SMA. In vivo, strong expression of URG11 was observed in the tubular epithelia of 5/6-nephrectomized rats, and a Western blot analysis demonstrated a close correlation between HIF-1{alpha} and URG11 protein levels. Altogether, our results indicate that URG11 mediates hypoxia-induced EMT through the suppression of E-cadherin and the activation of the {beta}-catenin/TCF pathway.

  17. Brain metastases from lung cancer show increased expression of DVL1, DVL3 and beta-catenin and down-regulation of E-cadherin.

    PubMed

    Kafka, Anja; Tomas, Davor; Beroš, Vili; Pećina, Hrvoje Ivan; Zeljko, Martina; Pećina-Šlaus, Nives

    2014-06-13

    The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p=0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p=0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.

  18. Brain Metastases from Lung Cancer Show Increased Expression of DVL1, DVL3 and Beta-Catenin and Down-Regulation of E-Cadherin

    PubMed Central

    Kafka, Anja; Tomas, Davor; Beroš, Vili; Pećina, Hrvoje Ivan; Zeljko, Martina; Pećina-Šlaus, Nives

    2014-01-01

    The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1), Dishevelled-3 (DVL3), E-cadherin (CDH1) and beta-catenin (CTNNB1). Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR)/loss of heterozygosity (LOH). Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p = 0.0001). Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC) were significantly associated to CDH1 LOH (p = 0.001). Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain. PMID:24933634

  19. EpCAM Intracellular Domain Promotes Porcine Cell Reprogramming by Upregulation of Pluripotent Gene Expression via Beta-catenin Signaling

    PubMed Central

    Yu, Tong; Ma, Yangyang; Wang, Huayan

    2017-01-01

    Previous study showed that expression of epithelial cell adhesion molecule (EpCAM) was significantly upregulated in porcine induced pluripotent stem cells (piPSCs). However, the regulatory mechanism and the downstream target genes of EpCAM were not well investigated. In this study, we found that EpCAM was undetectable in fibroblasts, but highly expressed in piPSCs. Promoter of EpCAM was upregulated by zygotic activated factors LIN28, and ESRRB, but repressed by maternal factors OCT4 and SOX2. Knocking down EpCAM by shRNA significantly reduced the pluripotent gene expression. Conversely, overexpression of EpCAM significantly increased the number of alkaline phosphatase positive colonies and elevated the expression of endogenous pluripotent genes. As a key surface-to-nucleus factor, EpCAM releases its intercellular domain (EpICD) by a two-step proteolytic processing sequentially. Blocking the proteolytic processing by inhibitors TAPI-1 and DAPT could reduce the intracellular level of EpICD and lower expressions of OCT4, SOX2, LIN28, and ESRRB. We noticed that increasing intracellular EpICD only was unable to improve activity of EpCAM targeted genes, but by blocking GSK-3 signaling and stabilizing beta-catenin signaling, EpICD could then significantly stimulate the promoter activity. These results showed that EpCAM intracellular domain required beta-catenin signaling to enhance porcine cell reprogramming. PMID:28393933

  20. Attenuated Response to Methamphetamine Sensitization and Deficits in Motor Learning and Memory after Selective Deletion of [beta]-Catenin in Dopamine Neurons

    ERIC Educational Resources Information Center

    Diaz-Ruiz, Oscar; Zhang, YaJun; Shan, Lufei; Malik, Nasir; Hoffman, Alexander F.; Ladenheim, Bruce; Cadet, Jean Lud; Lupica, Carl R.; Tagliaferro, Adriana; Brusco, Alicia; Backman, Cristina M.

    2012-01-01

    In the present study, we analyzed mice with a targeted deletion of [beta]-catenin in DA neurons (DA-[beta]cat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-[beta]cat KO mice showed significant…

  1. Soy isoflavone genistein upregulates epithelial adhesion molecule e-cadherin expression and attenuates beta-catenin signaling in mammary epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enhanced Wnt/beta -catenin signaling and loss of E-cadherin expression are considered hallmarks of mammary tumorigenesis. Mammary tumor protection by dietary intake of soy-rich foods and the soy isoflavone genistein (Gen) is widely regarded based on numerous epidemiological and animal studies; howev...

  2. Circadian rhythms, Wnt/beta-catenin pathway and PPAR alpha/gamma profiles in diseases with primary or secondary cardiac dysfunction

    PubMed Central

    Lecarpentier, Yves; Claes, Victor; Duthoit, Guillaume; Hébert, Jean-Louis

    2014-01-01

    Circadian clock mechanisms are far-from-equilibrium dissipative structures. Peroxisome proliferator-activated receptors (PPAR alpha, beta/delta, and gamma) play a key role in metabolic regulatory processes, particularly in heart muscle. Links between circadian rhythms (CRs) and PPARs have been established. Mammalian CRs involve at least two critical transcription factors, CLOCK and BMAL1 (Gekakis et al., 1998; Hogenesch et al., 1998). PPAR gamma plays a major role in both glucose and lipid metabolisms and presents circadian properties which coordinate the interplay between metabolism and CRs. PPAR gamma is a major component of the vascular clock. Vascular PPAR gamma is a peripheral regulator of cardiovascular rhythms controlling circadian variations in blood pressure and heart rate through BMAL1. We focused our review on diseases with abnormalities of CRs and with primary or secondary cardiac dysfunction. Moreover, these diseases presented changes in the Wnt/beta-catenin pathway and PPARs, according to two opposed profiles. Profile 1 was defined as follows: inactivation of the Wnt/beta-catenin pathway with increased expression of PPAR gamma. Profile 2 was defined as follows: activation of the Wnt/beta-catenin pathway with decreased expression of PPAR gamma. A typical profile 1 disease is arrhythmogenic right ventricular cardiomyopathy, a genetic cardiac disease which presents mutations of the desmosomal proteins and is mainly characterized by fatty acid accumulation in adult cardiomyocytes mainly in the right ventricle. The link between PPAR gamma dysfunction and desmosomal genetic mutations occurs via inactivation of the Wnt/beta-catenin pathway presenting oscillatory properties. A typical profile 2 disease is type 2 diabetes, with activation of the Wnt/beta-catenin pathway and decreased expression of PPAR gamma. CRs abnormalities are present in numerous pathologies such as cardiovascular diseases, sympathetic/parasympathetic dysfunction, hypertension, diabetes

  3. MENA is a transcriptional target of the Wnt/beta-catenin pathway.

    PubMed

    Najafov, Ayaz; Seker, Tuncay; Even, Ipek; Hoxhaj, Gerta; Selvi, Osman; Ozel, Duygu Esen; Koman, Ahmet; Birgül-İyison, Necla

    2012-01-01

    Wnt/β-catenin signalling pathway plays important roles in embryonic development and carcinogenesis. Overactivation of the pathway is one of the most common driving forces in major cancers such as colorectal and breast cancers. The downstream effectors of the pathway and its regulation of carcinogenesis and metastasis are still not very well understood. In this study, which was based on two genome-wide transcriptomics screens, we identify MENA (ENAH, Mammalian enabled homologue) as a novel transcriptional target of the Wnt/β-catenin signalling pathway. We show that the expression of MENA is upregulated upon overexpression of degradation-resistant β-catenin. Promoters of all mammalian MENA homologues contain putative binding sites for Tcf4 transcription factor--the primary effector of the Wnt/β-catenin pathway and we demonstrate functionality of these Tcf4-binding sites using luciferase reporter assays and overexpression of β-catenin, Tcf4 and dominant-negative Tcf4. In addition, lithium chloride-mediated inhibition of GSK3β also resulted in increase in MENA mRNA levels. Chromatin immunoprecipitation showed direct interaction between β-catenin and MENA promoter in Huh7 and HEK293 cells and also in mouse brain and liver tissues. Moreover, overexpression of Wnt1 and Wnt3a ligands increased MENA mRNA levels. Additionally, knock-down of MENA ortholog in D. melanogaster eyeful and sensitized eye cancer fly models resulted in increased tumor and metastasis formations. In summary, our study identifies MENA as novel nexus for the Wnt/β-catenin and the Notch signalling cascades.

  4. E-cadherin and beta-catenin expression in Epstein-Barr virus-associated gastric carcinoma and their prognostic significance

    PubMed Central

    Koriyama, Chihaya; Akiba, Suminori; Itoh, Tetsuhiko; Sueyoshi, Kazunobu; Minakami, Yoshie; Corvalan, Alejandro; Yonezawa, Suguru; Eizuru, Yoshito

    2007-01-01

    AIM: To examine the role of E-cadherin and beta-catenin in carcinogenesis and to assess their prognostic implication in Epstein-Barr virus-associated gastric carcinomas (EBV-GCs). METHODS: We compared the frequency of E-cadherin and beta-catenin expression in 59 EBV-GCs and 120 non-EBV-GCs, and examined the association between patients' prognosis and the expressions of these proteins. RESULTS: Neither the cellular-membranous nor the cytoplasmic E-cadherin expression showed any difference between EBV-GCs and non-EBV-GCs. On the other hand, loss of membranous expression of beta-catenin occurred more frequently in non-EBV-GCs than EBV-GCs [odds ratio = 0.41; 95% confidence interval (CI), 0.19-0.90]. Furthermore, the nuclear and/or cytoplosmic expression of beta-catenin was seen more frequently in EBV-GCs than non-EBV-GCs (odds ratio = 2.23; 95% CI, 0.97-5.09), and was observed in a larger proportion of carcinoma cells of EBV-GCs than non-EBV-GCs (P = 0.024). Survival analysis for non-EBV-GC revealed that lymph node metastasis was significantly associated with poor prognosis (P < 0.001). Among EBV-GCs, the depth of invasion (P = 0.005), lymph node metastasis (P = 0.004) and an intestinal type by Lauren classification (hazard ratio = 9.47; 95% CI, 2.67-33.6) were significantly associated with poor prognosis. On the other hand, nuclear and/or cytoplasmic expression of beta-catenin was associated with a better prognosis in patients with EBV-GC (hazard ratio = 0.32; 95% CI, 0.11-0.93). CONCLUSION: We observed more frequent preservation of beta-catenin in cell membrane and accumulation in nuclei and/or cytoplasm in EBV-GCs than in non-EBV-GCs. Factors involved in the prognosis of EBV-GCs and non-EBV-GCs are different in the two conditions. PMID:17663505

  5. Selection of multipotent stem cells during morphogenesis of small intestinal crypts of Lieberkuhn is perturbed by stimulation of Lef-1/beta-catenin signaling.

    PubMed

    Wong, Melissa H; Huelsken, Joerg; Birchmeier, Walter; Gordon, Jeffrey I

    2002-05-03

    Studies of chimeric mice have disclosed that the stem cell hierarchy in the small intestinal epithelium is established during formation of its proliferative units (crypts of Lieberkühn). This process involves a selection among several multipotential progenitors so that ultimately only one survives to supply descendants to the fully formed crypt. In this report, we examine the hypothesis that the level of beta-catenin (beta-cat)-mediated signaling is an important factor regulating this stem cell selection. In the canonical Wnt signaling pathway, beta-catenin can partner with Lef-1/Tcf high mobility group (HMG) box transcription factors to control gene expression. Both Lef-1 and Tcf-4 mRNAs are produced in the fetal mouse small intestine. Tcf-4 expression is sustained, whereas Lef-1 levels fall as crypt formation is completed during the first two postnatal weeks. A Tcf-4 gene knockout is known to block intestinal epithelial proliferation in late fetal life. Therefore, to test the hypothesis, we enhanced beta-catenin signaling in a chimeric mouse model in which the stem cell selection could be monitored. A fusion protein containing the HMG box domain of Lef-1 linked to the trans-activation domain of beta-catenin (Lef-1/beta-cat) was constructed to promote direct stimulation of signaling without being retained in the cytoplasm through interactions with E-cadherin and Apc/Axin. Lef-1/beta-cat was expressed in 129/Sv embryonic stem cell-derived small intestinal epithelial progenitors present in developing B6-ROSA26<-->129/Sv chimeras. Lef-1/beta-cat stimulated expression of a known beta-catenin target (E-cadherin), suppressed expression of Apc and Axin, and induced apoptosis in 129/Sv but not in neighboring B6-ROSA26 epithelial cells. This apoptotic response was not associated with any detectable changes in cell division within the Lef-1/beta-cat-expressing epithelium. By the time crypt development was completed, all 129/Sv epithelial cells were lost. These results

  6. P53 levels determine outcome during beta-catenin tumor initiation and metastasis in the mammary gland and male germ cells.

    PubMed

    Ridgeway, A G; McMenamin, J; Leder, P

    2006-06-15

    beta-Catenin, an oncogene, and P53, a tumor suppressor, are common targets of mutation in human cancers. It has been observed that P53 is often inactivated in tumors involving beta-catenin activation. In an attempt to model this situation in vivo, we crossed the previously characterized MMTV-DeltaN-beta-catenin mouse with the P53 knockout mouse. Female multiparous mice that carry the MMTV-DeltaN-beta-catenin transgene and that are heterozygous for P53 (Tg(DeltaN-betaCat)/+, P53+/-) display an increased tumor burden (2.05 vs 1.31 tumors/animal), with a generally more advanced pathology, and increased metastatic rate (39 vs 0%) relative to transgenic female mice that are wild type for P53 (Tg(DeltaN-betaCat)/+, P53+/+). These differences were not due to complete loss of P53 as only one of 21 tumors demonstrated loss of heterozygosity at the P53 locus. Furthermore, no mutations were present in tumors retaining a single wild-type allele. Tg(DeltaN-betaCat)/+, P53-/- male mice developed testicular teratomas and survived an average of 65 days, whereas non-Tg(DeltaN-betaCat), P53-/- males survived an average of 84 days. Sixty-two percent of Tg(DeltaN-betaCat), P53-/- mice developed testicular teratomas, whereas only 10% of the non-Tg(DeltaN-betaCat), P53-/- mice developed these tumors. These results indicate that the level of P53 and the tissue of origin are important factors in determining outcome of cancer caused by oncogene activation.

  7. Discovery of Novel Drugs To Improve Bone Health in Neurofibromatosis Type 1: The Wnt/Beta-Catenin Pathway in Fracture Repair and Pseudarthrosis

    DTIC Science & Technology

    2014-06-01

    Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Patients with Neurofibromatosis ( NF1 ) exhibit deficient...bone healing/ The cause of poor bone healing in NF1 is unclear, and pharmacologic approaches to improve bone repair are lacking. Beta-catenin is a...undifferentiated fibroblast-like cells persist at the fracture site, resulting in a pseudarthrosis. Genetically engineered mice in which the Nf1 gene can be

  8. Critical interactions between TGF-beta signaling/ELF, and E-cadherin/beta-catenin mediated tumor suppression.

    PubMed

    Katuri, V; Tang, Y; Li, C; Jogunoori, W; Deng, C-X; Rashid, A; Sidawy, A N; Evans, S; Reddy, E P; Mishra, B; Mishra, L

    2006-03-23

    Inactivation of the transforming growth factor-beta (TGF-beta) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/- and elf+/- / Smad4+/- mutant mice. We found that embryonic liver fodrin (ELF), a beta-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell-cell contacts and E-cadherin-beta-catenin-dependent epithelial cell-cell adhesion is disrupted in elf+/- / Smad4+/- mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-beta stimulation. In contrast, elf+/- / Smad4+/- mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-beta signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.

  9. beta-catenin is strongly elevated in rat colonic epithelium following short-term intermittent treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and a high-fat diet.

    PubMed

    Wang, Rong; Dashwood, W Mohaiza; Löhr, Christiane V; Fischer, Kay A; Nakagama, Hitoshi; Williams, David E; Dashwood, Roderick H

    2008-09-01

    Colon tumors expressing high levels of beta-catenin and c-myc have been reported in male F344 rats given three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) alternating with a high-fat (HF) diet. Using the same experimental protocol, rats were euthanized 24 h after the last dose of PhIP so as to examine early changes in colonic crypt homeostasis and beta-catenin expression, before the onset of frank tumors. PhIP/HF dosing caused a significant increase in the bromodeoxyuridine labeling index throughout the entire colon, and within the colonic crypt column cleaved caspase-3 was elevated in the basal and central zones, but reduced in the luminal region. In vehicle/HF controls, beta-catenin was immunolocalized primarily at the border between cells at the top of the crypt, whereas in rats given PhIP/HF diet there was strong cytoplasmic staining, which appeared as a gradient of increased beta-catenin extending from the base of the crypt column to the luminal region. Quantitative real-time PCR and immunoblot analyses confirmed that beta-catenin and c-myc were increased significantly in the colonic mucosa of rats given PhIP/HF diet. Collectively, these findings suggest that PhIP/HF cycling alters beta-catenin and c-myc expression in the colonic mucosa, resulting in expansion of the proliferative zone and redistribution of apoptotic cells from the lumen to the central and basal regions of the colonic crypt. Thus, during the early stages of colon carcinogenesis, alternating exposure to heterocyclic amines and a high-fat diet might facilitate molecular changes resulting in dysregulated beta-catenin and c-myc expression.

  10. Regulation of AR and (Beta)-Catenin Signaling by Pin 1 in Prostate Cancer

    DTIC Science & Technology

    2005-10-01

    function. Nature 406:1009-1012. 41. Rubinfeld, B., I. Albert , E. Porfiri, C. Fiol, S. Munemitsu, and P. Polakis. 1996. Binding of GSK3beta to the APC...Hurlstone, H. K. van der, E. Batlle , D. Coudreuse, A. P. Haramis, M. Tjon-Pon-Fong, P. Moerer, B. M. van den, G Soete, S. Pals, M. Eilers, R. Medema

  11. PTEN Regulates Beta-Catenin in Androgen Signaling: Implication in Prostate Cancer Progression

    DTIC Science & Technology

    2006-03-01

    Leland Stanford Junior University Stanford, California 94305-4125...DAMD17-03-1-0090 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Leland Stanford Junior University Stanford, California...testis. Mol Endocrinol 14:14–26 4. Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, Shuai K 1997 Specific inhibition of Stat3 signal transduction by

  12. PTEN Regulates Beta-Catenin in Androgen Signaling: Implication in Prostate Cancer Progression

    DTIC Science & Technology

    2005-03-01

    modulation of the PI3K/Akt downstream effector, GSK3 3 (glycogen synthase kinase 3 3). Its inactivation by phosphorylation results in increased nuclear...ng 30 ng MNormal IgG - + Figure 3. Overexpression of IGF-1 receptors enhances AR-mediated transcription. (A) Transient transfections were performed in...Ruel, L. and Woodgett, J.R. (1996) Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol, 6

  13. Modulation of Beta-Catenin Activity with PKD1 in Prostate Cancer

    DTIC Science & Technology

    2011-04-01

    inhibits benzo [ a ] pyrene induced upregulation of HPV E7. Molecular Carcinogenesis 2011;50(1):47-57. 4. Yallapu MM., Othman SF., Curtis ET., Gupta B...law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB...involved in interaction and modulation of β-catenin activity. The retroviral system is a well established technique that yields much higher

  14. CB1 cannabinoid receptors increase neuronal precursor proliferation through AKT/glycogen synthase kinase-3beta/beta-catenin signaling.

    PubMed

    Trazzi, Stefania; Steger, Martin; Mitrugno, Valentina Maria; Bartesaghi, Renata; Ciani, Elisabetta

    2010-03-26

    The endocannabinoid system is involved in the regulation of many physiological effects in the central and peripheral nervous system. Recent findings have demonstrated the presence of a functional endocannabinoid system within neuronal progenitors located in the hippocampus and ventricular/subventricular zone that participates in the regulation of cell proliferation. It is presently unknown whether the endocannabinoid system exerts a widespread effect on neuronal precursors from different neurogenic regions, and very little is known about the signaling by which it regulates neuronal precursor proliferation. Herein, we demonstrate the presence of cannabinoid CB(1) receptors in granule cell precursors (GCPs) during early cerebellar development. Activation of CB(1) receptors by HU-210 promoted GCP proliferation in vitro, an effect that was prevented by a selective CB(1) antagonist. Accordingly, in vivo experiments showed that GCP proliferation was increased by chronic HU-210 treatment and that in CB(1)-deficient mice cell proliferation was significantly lower than in wild-type littermates, indicating that the endocannabinoid system is physiologically involved in regulation of GCP proliferation. The pro-proliferative effect of cannabinoids in GCPs was mediated through the CB(1)/AKT/glycogen synthase kinase-3beta/beta-catenin pathway. Involvement of this pathway was also observed in cultures of neuronal precursors from the subventricular zone, suggesting that this pathway may be a general mechanism by which endocannabinoids regulate proliferation of neuronal precursors. These observations suggest that endocannabinoids constitute a new family of lipid signaling cues that may exert a widespread effect on neuronal precursor proliferation during brain development.

  15. Dpr Acts as a molecular switch, inhibiting Wnt signaling when unphosphorylated, but promoting Wnt signaling when phosphorylated by casein kinase Idelta/epsilon.

    PubMed

    Teran, Evelyn; Branscomb, Aron D; Seeling, Joni M

    2009-01-01

    The Wnt pathway is a key regulator of development and tumorigenesis. Dpr (Dact/Frodo) influences Wnt signaling in part through the interaction of its PDZ-B domain with Dsh's PDZ domain. Studies have shown that XDpr1a and its close relative, Frodo, are involved in multiple steps of the Wnt pathway in either inhibitory or activating roles. We found that XDpr1a is phosphorylated by casein kinase Idelta/epsilon (CKIdelta/epsilon), an activator of Wnt signaling, in the presence of XDsh. Abrogating XDpr1a's ability to bind XDsh through mutation of XDpr1a's PDZ-B domain blocks CK1delta/epsilon's phosphorylation of XDpr1a. Conversely, XDsh possessing a mutation in its PDZ domain that is unable to bind XDpr1a does not promote XDpr1a phosphorylation. Phosphorylation of XDpr1a and XDsh by CKIdelta/epsilon decreases their interaction. Moreover, the phosphorylation of XDpr1a by CKIdelta/epsilon not only abrogates XDpr1a's promotion of beta-catenin degradation but blocks beta-catenin degradation. Our data suggest that XDpr1a phosphorylation by CKIdelta/epsilon is dependent on the interaction of XDpr1a's PDZ-B domain with XDsh's PDZ domain, and that the phosphorylation state of XDpr1a determines whether it inhibits or activates Wnt signaling.

  16. Chondroitin Sulfate-E Is a Negative Regulator of a Pro-Tumorigenic Wnt/Beta-Catenin-Collagen 1 Axis in Breast Cancer Cells

    PubMed Central

    Willis, Catherine M.; Klüppel, Michael

    2014-01-01

    Expression of the glycosaminoglycan chondroitin sulfate-E (CS-E) is misregulated in many human cancers, including breast cancer. Cell-surface associated CS-E has been shown to have pro-tumorigenic functions, and pharmacological treatment with exogenous CS-E has been proposed to interfere with tumor progression mediated by endogenous CS-E. However, the effects of exogenous CS-E on breast cancer cell behavior, and the molecular mechanisms deployed by CS-E are not well understood. We show here that treatment with CS-E, but not other chondroitin forms, could interfere with the invasive protrusion formation and migration of breast cancer cells in three-dimensional organotypic cultures. Microarray analysis identified transcriptional programs controlled by CS-E in these cells. Importantly, negative regulation of the pro-metastatic extracellular matrix gene Col1a1 was required for the anti-migratory effects of exogenous CS-E. Knock-down of Col1a1 gene expression mimics the effects of CS-E treatment, while exposing cells to a preformed collagen I matrix interfered with the anti-migratory effects of CS-E. In addition, CS-E specifically interfered with Wnt/beta-catenin signaling, a known pro-tumorigenic pathway. Lastly, we demonstrate that Col1a1 is a positively regulated target gene of the Wnt/beta-catenin pathway in breast cancer cells. Together, our data identify treatment with exogenous CS-E as negative regulatory mechanism of breast cancer cell motility through interference with a pro-tumorigenic Wnt/beta-catenin - Collagen I axis. PMID:25090092

  17. The expression of syndecan-1 and -2 is associated with Gleason score and epithelial-mesenchymal transition markers, E-cadherin and beta-catenin, in prostate cancer.

    PubMed

    Contreras, Hector R; Ledezma, Rodrigo A; Vergara, Jorge; Cifuentes, Federico; Barra, Cristina; Cabello, Pablo; Gallegos, Ivan; Morales, Bernardo; Huidobro, Christian; Castellón, Enrique A

    2010-01-01

    The epithelial-mesenchymal transition (EMT) is considered a key step in tumor progression, where the invasive cancer cells change from epithelial to mesenchymal phenotype. During this process, a decrease or loss in adhesion molecules expression and an increase in migration molecules expression are observed. The aim of this work was to determine the expression and cellular distribution of syndecan-1 and -2 (migration molecules) and E-cadherin and beta-catenin (adhesion molecules) in different stages of prostate cancer progression. A quantitative immunohistochemical study of these molecules was carried out in tissue samples from benign prostatic hyperplasia and prostate carcinoma, with low and high Gleason score, obtained from biopsies archives of the Clinic Hospital of the University of Chile and Dipreca Hospital. Polyclonal specific antibodies and amplification system of estreptavidin-biotin peroxidase and diaminobenzidine were used. Syndecan-1 was uniformly expressed in basolateral membranes of normal epithelium, changing to a granular cytoplasmatic expression pattern in carcinomas. Syndecan-2 was observed mainly in a cytoplasmatic granular pattern, with high immunostaining intensity in areas of low Gleason score. E-cadherin was detected in basolateral membrane of normal epithelia showing decreased expression in high Gleason score samples. beta-Catenin was found in cell membranes of normal epithelia changing its distribution toward the nucleus and cytoplasm in carcinoma samples. We concluded that changes in expression and cell distribution of E-cadherin and beta-catenin correlated with the progression degree of prostate adenocarcinoma, suggesting a role of these molecules as markers of progression and prognosis. Furthermore, changes in the pattern expression of syndecan-1 and -2 indicate that both molecules may be involved in the EMT and tumor progression of prostate cancer.

  18. Novel Cancer Chemotherapy Hits by Molecular Topology: Dual Akt and Beta-Catenin Inhibitors

    PubMed Central

    Morell, Cecilia; Rodríguez-Henche, Nieves; Recio-Iglesias, Maria Carmen; Garcia-Domenech, Ramon

    2015-01-01

    Background and Purpose Colorectal and prostate cancers are two of the most common types and cause of a high rate of deaths worldwide. Therefore, any strategy to stop or at least slacken the development and progression of malignant cells is an important therapeutic choice. The aim of the present work is the identification of novel cancer chemotherapy agents. Nowadays, many different drug discovery approaches are available, but this paper focuses on Molecular Topology, which has already demonstrated its extraordinary efficacy in this field, particularly in the identification of new hit and lead compounds against cancer. This methodology uses the graph theoretical formalism to numerically characterize molecular structures through the so called topological indices. Once obtained a specific framework, it allows the construction of complex mathematical models that can be used to predict physical, chemical or biological properties of compounds. In addition, Molecular Topology is highly efficient in selecting and designing new hit and lead drugs. According to the aforementioned, Molecular Topology has been applied here for the construction of specific Akt/mTOR and β-catenin inhibition mathematical models in order to identify and select novel antitumor agents. Experimental Approach Based on the results obtained by the selected mathematical models, six novel potential inhibitors of the Akt/mTOR and β-catenin pathways were identified. These compounds were then tested in vitro to confirm their biological activity. Conclusion and Implications Five of the selected compounds, CAS n° 256378-54-8 (Inhibitor n°1), 663203-38-1 (Inhibitor n°2), 247079-73-8 (Inhibitor n°3), 689769-86-6 (Inhibitor n°4) and 431925-096 (Inhibitor n°6) gave positive responses and resulted to be active for Akt/mTOR and/or β-catenin inhibition. This study confirms once again the Molecular Topology’s reliability and efficacy to find out novel drugs in the field of cancer. PMID:25910265

  19. Promotion versus suppression of rat colon carcinogenesis by chlorophyllin and chlorophyll: modulation of apoptosis, cell proliferation, and beta-catenin/Tcf signaling.

    PubMed

    Blum, Carmen A; Xu, Meirong; Orner, Gayle A; Darío Díaz, G; Li, Qingjie; Dashwood, Wan Mohaiza; Bailey, George S; Dashwood, Roderick H

    2003-01-01

    The carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 1,2-dimethylhydrazine (DMH) induce colon tumors in the rat that contain mutations in beta-catenin, but the mutation pattern can be influenced by exposure to dietary phytochemicals, such as the water-soluble derivative of chlorophyll called chlorophyllin. Whereas chlorophyllin is an effective blocking agent during the initiation phase, post-initiation responses depend upon the exposure protocol, and can be influenced by the initiating agent and the concentration of chlorophyllin. Post-initiation treatment with 0.001% chlorophyllin (w/v) in the drinking water promoted colon carcinogenesis in the rat, but much higher concentrations (1.0% chlorophyllin) led to suppression. Bromodeoxyuridine and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) indices revealed that the promotional concentration of 0.001% chlorophyllin increased the ratio of cell proliferation to apoptosis in the colonic crypts, whereas concentrations in the range 0.0l-1.0% chlorophyllin modestly reduced this ratio. Molecular studies showed that the spectrum of beta-catenin mutations was markedly different in chlorophyllin-promoted colon tumors--many of the mutations led to direct substitutions of critical Ser/Thr residues within the glycogen synthase kinase-3beta (GSK-3beta) region, whereas in all other groups, including DMH and IQ controls, the mutations typically affected amino acids adjacent to Ser(33). Substitution of critical Ser/Thr residues caused beta-catenin and c-Jun proteins to be markedly over-expressed compared with tumors in which the mutations substituted amino acid residues flanking these critical Ser/Thr sites. In a separate study, rats were exposed to IQ or azoxymethane (AOM), a metabolite of DMH, and they were treated post-initiation with chlorophyllin, chlorophyll, copper, or phytol in the diet. Natural chlorophyll (0.08%) suppressed AOM- and IQ-induced aberrant crypt foci (ACF), whereas

  20. Monitoring Interactions and Dynamics of Endogenous Beta-catenin With Intracellular Nanobodies in Living Cells*

    PubMed Central

    Traenkle, Bjoern; Emele, Felix; Anton, Roman; Poetz, Oliver; Haeussler, Ragna S.; Maier, Julia; Kaiser, Philipp D.; Scholz, Armin M.; Nueske, Stefan; Buchfellner, Andrea; Romer, Tina; Rothbauer, Ulrich

    2015-01-01

    β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays. PMID:25595278

  1. Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling.

    PubMed

    Zemans, Rachel L; Briones, Natalie; Campbell, Megan; McClendon, Jazalle; Young, Scott K; Suzuki, Tomoko; Yang, Ivana V; De Langhe, Stijn; Reynolds, Susan D; Mason, Robert J; Kahn, Michael; Henson, Peter M; Colgan, Sean P; Downey, Gregory P

    2011-09-20

    Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real-time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of β-catenin signaling in response to neutrophil transmigration. Knockdown of β-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of β-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of β-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that β-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

  2. Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair.

    PubMed

    Baht, Gurpreet S; Nadesan, Puviindran; Silkstone, David; Alman, Benjamin A

    2017-02-22

    Patients with Neurofibromatosis type 1 display delayed fracture healing and the increased deposition of fibrous tissue at the fracture site. Severe cases can lead to non-union and even congenital pseudarthrosis. Neurofibromatosis type 1 is caused by a mutation in the NF1 gene and mice lacking the Nf1 gene show a fracture repair phenotype similar to that seen in patients. Tissue from the fracture site of patients with Neurofibromatosis type 1 and from mice deficient in the Nf1 gene both show elevated levels of β-catenin protein and activation of β-catenin mediated signaling. Constitutively elevated β-catenin leads to a delayed and fibrous fracture repair process, and (RS)-5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine (Nefopam, a centrally-acting, non-narcotic analgesic agent) inhibits β-catenin mediated signaling during skin wound repair. Here we investigate Nefopam's potential as a modulator of bone repair in mice deficient in Nf1. Mice were treated with Nefopam and investigated for bone fracture repair. Bone marrow stromal cells flushed from the long bones of unfractured mice were treated with Nefopam and investigated for osteogenic potential. Treatment with Nefopam was able to lower the β-catenin level and the Axin2 transcript level in the fracture calluses of Nf1 deficient mice. Cultures from the bone marrow of Nf1(-/-) mice had significantly lower osteoblastic colonies and mineralized nodules, which was increased when cells were cultured in the presence of Nefopam. Fracture calluses were harvested and analyzed 14days and 21days after injury. Nf1(-/-) calluses had less bone, less cartilage, and higher fibrous tissue content than control calluses. Treatment with Nefopam increased the bone and cartilage content and decreased the fibrous tissue content in Nf1(-/-) calluses. These findings present a potential treatment for patients with Neurofibromatosis 1 in the context of bone repair. Since Nefopam is already in use in patient care, it could be

  3. Mucin-depleted foci have beta-catenin gene mutations, altered expression of its protein, and are dose- and time-dependent in the colon of 1,2-dimethylhydrazine-treated rats.

    PubMed

    Femia, Angelo Pietro; Bendinelli, Benedetta; Giannini, Augusto; Salvadori, Maddalena; Pinzani, Pamela; Dolara, Piero; Caderni, Giovanna

    2005-08-10

    Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.

  4. Role of Akt/GSK-3beta/beta-catenin transduction pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-treated rats.

    PubMed

    Schakman, O; Kalista, S; Bertrand, L; Lause, P; Verniers, J; Ketelslegers, J M; Thissen, J P

    2008-08-01

    Decrease of muscle IGF-I plays a critical role in muscle atrophy caused by glucocorticoids (GCs) because IGF-I gene electrotransfer prevents muscle atrophy caused by GCs. The goal of the present study was to identify the intracellular mediators responsible for the IGF-I anti-atrophic action in GC-induced muscle atrophy. We first assessed the IGF-I transduction pathway alterations caused by GC administration and their reversibility by local IGF-I overexpression performed by electrotransfer. Muscle atrophy induced by dexamethasone (dexa) administration occurred with a decrease in Akt (-53%; P<0.01) phosphorylation together with a decrease in beta-catenin protein levels (-40%; P<0.001). Prevention of atrophy by IGF-I was associated with restoration of Akt phosphorylation and beta-catenin levels. We then investigated whether muscle overexpression of these intracellular mediators could mimic the IGF-I anti-atrophic effects. Overexpression of a constitutively active form of Akt induced a marked fiber hypertrophy in dexa-treated animals (+175% of cross-sectional area; P<0.001) and prevented dexa-induced atrophy. This hypertrophy was associated with an increase in phosphorylated GSK-3beta (+17%; P<0.05) and in beta-catenin content (+35%; P<0.05). Furthermore, overexpression of a dominant-negative GSK-3beta or a stable form of beta-catenin increased fiber cross-sectional area by, respectively, 23% (P<0.001) and 29% (P<0.001) in dexa-treated rats, preventing completely the atrophic effect of GC. In conclusion, this work indicates that Akt, GSK-3beta, and beta-catenin probably contribute together to the IGF-I anti-atrophic effect in GC-induced muscle atrophy.

  5. High molecular weight polysaccharide that binds and inhibits virus

    DOEpatents

    Konowalchuk, Thomas W

    2014-01-14

    This invention provides a high molecular weight polysaccharide capable of binding to and inhibiting virus and related pharmaceutical formulations and methods on inhibiting viral infectivity and/or pathogenicity, as well as immunogenic compositions. The invention further methods of inhibiting the growth of cancer cells and of ameliorating a symptom of aging. Additionally, the invention provides methods of detecting and/or quantifying and/or isolating viruses.

  6. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    NASA Astrophysics Data System (ADS)

    Lee, L. S.

    1981-02-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

  7. Ghrelin Increases Beta-Catenin Level through Protein Kinase A Activation and Regulates OPG Expression in Rat Primary Osteoblasts

    PubMed Central

    Mrak, Emanuela; Casati, Lavinia; Pagani, Francesca; Rubinacci, Alessandro; Zarattini, Guido; Sibilia, Valeria

    2015-01-01

    Ghrelin, by binding growth hormone secretagogue receptor (GHS-R), promotes osteoblast proliferation but the signaling mechanism of GHS-R on these cells remains unclear. Since canonical Wnt/β-catenin pathway is critically associated with bone homeostasis, we investigated its involvement in mediating ghrelin effects in osteoblasts and in osteoblast-osteoclast cross talk. Ghrelin (10−10M) significantly increased β-catenin levels in rat osteoblasts (rOB). This stimulatory action on β-catenin involves a specific interaction with GHS-R1a, as it is prevented by the selective GHS-R1a antagonist, D-Lys3-GHRP-6 (10−7M). The effect of ghrelin on β-catenin involves the phosphorylation and inactivation of GSK-3β via protein kinase A (PKA). Inhibition of PKA activity reduces the facilitatory action of ghrelin on β-catenin stabilization. Ghrelin treatment of rOB significantly increases the expression of osteoprotegerin (OPG), which plays an important role in the regulation of osteoclastogenesis, and this effect is blocked by D-Lys3-GHRP-6. Furthermore, ghrelin reduced RANKL/OPG ratio thus contrasting osteoclastogenesis. Accordingly, conditioned media from rOB treated with ghrelin decreased the number of multinucleated TRAcP+ cells as compared with the conditioned media from untreated-control rOB. Our data suggest new roles for ghrelin in modulating bone homeostasis via a specific interaction with GHSR-1a in osteoblasts with subsequent enhancement of both β-catenin levels and OPG expression. PMID:25866509

  8. Bilirubin Binding to PPARα Inhibits Lipid Accumulation

    PubMed Central

    Stec, David E.; John, Kezia; Trabbic, Christopher J.; Luniwal, Amarjit; Hankins, Michael W.; Baum, Justin

    2016-01-01

    Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin. PMID:27071062

  9. Bilirubin Binding to PPARα Inhibits Lipid Accumulation.

    PubMed

    Stec, David E; John, Kezia; Trabbic, Christopher J; Luniwal, Amarjit; Hankins, Michael W; Baum, Justin; Hinds, Terry D

    2016-01-01

    Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin.

  10. Inhibition Of Call-Cell Binding By Kipid Assemblies

    DOEpatents

    Nagy, Jon O. , Bargatze, Robert F.

    2003-12-16

    This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

  11. Inhibition of cell-cell binding by lipid assemblies

    DOEpatents

    Nagy, Jon O.; Bargatze, Robert F.

    2001-05-22

    This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

  12. Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

    PubMed

    Sage, Jay M; Cura, Anthony J; Lloyd, Kenneth P; Carruthers, Anthony

    2015-05-15

    Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

  13. Inhibition of tobramycin diffusion by binding to alginate.

    PubMed Central

    Nichols, W W; Dorrington, S M; Slack, M P; Walmsley, H L

    1988-01-01

    [3H]tobramycin bound to sodium alginate and to exopolysaccharide prepared from two mucoid strains of Pseudomonas aeruginosa. Binding to sodium alginate was similar to binding to exopolysaccharide, both in the dependence on tobramycin concentration and in the maximum binding observed at saturation. Incorporation of sodium alginate into agar plates reduced the zone sizes of growth inhibition caused by tobramycin. The reductions in zone sizes were quantitatively accounted for by the binding of tobramycin to sodium alginate during diffusion of the antibiotic away from the well in which it had been placed at the start of the experiment. However, the binding of tobramycin to the exopolysaccharide of P. aeruginosa, and the resulting inhibition of diffusion of the antibiotic, did not significantly increase the penetration time of a spherical microcolony with a radius of 125 micron, such as might be found in the respiratory tract of a patient with cystic fibrosis (from a 90% penetration time of 12 s in the absence of exopolysaccharide to one of 35 s with an exopolysaccharide concentration of 1.0% [wt/vol]). PMID:3132093

  14. Inhibition of tobramycin diffusion by binding to alginate

    SciTech Connect

    Nichols, W.W.; Dorrington, S.M.; Slack, M.P.; Walmsley, H.L.

    1988-04-01

    (/sup 3/H)tobramycin bound to sodium alginate and to exopolysaccharide prepared from two mucoid strains of Pseudomonas aeruginosa. Binding to sodium alginate was similar to binding to exopolysaccharide, both in the dependence on tobramycin concentration and in the maximum binding observed at saturation. Incorporation of sodium alginate into agar plates reduced the zone sizes of growth inhibition caused by tobramycin. The reductions in zone sizes were quantitatively accounted for by the binding of tobramycin to sodium alginate during diffusion of the antibiotic away from the well in which it had been placed at the start of the experiment. However, the binding of tobramycin to the exopolysaccharide of P. aeruginosa, and the resulting inhibition of diffusion of the antibiotic, did not significantly increase the penetration time of a spherical microcolony with a radius of 125 micron, such as might be found in the respiratory tract of a patient with cystic fibrosis (from a 90% penetration time of 12 s in the absence of exopolysaccharide to one of 35 s with an exopolysaccharide concentration of 1.0% (wt/vol)).

  15. IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

    PubMed

    Ueno, Koji; Hirata, Hiroshi; Majid, Shahana; Tabatabai, Z Laura; Hinoda, Yuji; Dahiya, Rajvir

    2011-11-15

    The Wnt/β-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ β-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and β-catenin expression was increased. Also the β-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis.

  16. Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/beta-catenin signaling.

    PubMed

    Scholer-Dahirel, Alix; Schlabach, Michael R; Loo, Alice; Bagdasarian, Linda; Meyer, Ronald; Guo, Ribo; Woolfenden, Steve; Yu, Kristine K; Markovits, Judit; Killary, Karen; Sonkin, Dmitry; Yao, Yung-Mae; Warmuth, Markus; Sellers, William R; Schlegel, Robert; Stegmeier, Frank; Mosher, Rebecca E; McLaughlin, Margaret E

    2011-10-11

    Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.

  17. Binding Kinetics versus Affinities in BRD4 Inhibition.

    PubMed

    Kuang, Ming; Zhou, Jingwei; Wang, Laiyou; Liu, Zhihong; Guo, Jiao; Wu, Ruibo

    2015-09-28

    Bromodomains (BRDs) are protein modules that selectively recognize histones as a "reader" by binding to an acetylated lysine substrate. The human BRD4 has emerged as a promising drug target for a number of disease pathways, and several potent BRD inhibitors have been discovered experimentally recently. However, the detailed inhibition mechanism especially for the inhibitor binding kinetics is not clear. Herein, by employing classical molecular dynamics (MD) and state-of-the-art density functional QM/MM MD simulations, the dynamic characteristics of ZA-loop in BRD4 are revealed. And then the correlation between binding pocket size and ZA-loop motion is elucidated. Moreover, our simulations found that the compound (-)-JQ1 could be accommodated reasonably in thermodynamics whereas it is infeasible in binding kinetics against BRD4. Its racemate (+)-JQ1 proved to be both thermodynamically reasonable and kinetically achievable against BRD4, which could explain the previous experimental results that (+)-JQ1 shows a high inhibitory effect toward BRD4 (IC50 is 77 nM) while (-)-JQ1 is inactive (>10 μM). Furthermore, the L92/L94/Y97 in the ZA-loop and Asn140 in the BC-loop are identified to be critical residues in (+)-JQ1 binding/releasing kinetics. All these findings shed light on further selective inhibitor design toward BRD family, by exploiting the non-negligible ligand binding kinetics features and flexible ZA-loop motions of BRD, instead of only the static ligand-protein binding affinity.

  18. Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling.

    PubMed

    Li, Jifen; Swope, David; Raess, Natalia; Cheng, Lan; Muller, Eliane J; Radice, Glenn L

    2011-03-01

    Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.

  19. A novel LZAP-binding protein, NLBP, inhibits cell invasion.

    PubMed

    Kwon, Junhye; Cho, Hyun Jung; Han, Seung Hun; No, Jin Gu; Kwon, Jae Young; Kim, Hongtae

    2010-04-16

    LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-kappaB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-kappaB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-kappaB signaling, and increasing stability of the LZAP protein.

  20. A Novel LZAP-binding Protein, NLBP, Inhibits Cell Invasion*

    PubMed Central

    Kwon, Junhye; Cho, Hyun Jung; Han, Seung Hun; No, Jin Gu; Kwon, Jae Young; Kim, Hongtae

    2010-01-01

    LXXLL/leucine zipper-containing alternative reading frame (ARF)-binding protein (LZAP) was recently shown to function as a tumor suppressor through inhibition of the NF-κB signaling pathway. LZAP is also known as a negative regulator of cell invasion, and its expression was demonstrated to be reduced in several tumor tissues. However, the molecular mechanism of the negative effect of LZAP on cell invasion is unclear. In this study, we identify NLBP as a novel LZAP-binding protein using tandem affinity purification. We demonstrate the negative effects of NLBP on cell invasion and the NF-κB signaling pathway. NLBP expression was not detected in hepatocellular carcinoma cells with strong invasive activity, whereas its expression was detected in a hepatocellular carcinoma cell line with no invasive activity. We also demonstrate that these two proteins mutually affect the stability of each other by inhibiting ubiquitination of the other protein. Based on these results, we suggest that NLBP may act as a novel tumor suppressor by inhibiting cell invasion, blocking NF-κB signaling, and increasing stability of the LZAP protein. PMID:20164180

  1. Inhibition of oxytocin receptor function by direct binding of progesterone.

    PubMed

    Grazzini, E; Guillon, G; Mouillac, B; Zingg, H H

    1998-04-02

    The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5beta-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

  2. Inhibition of Midkine Augments Osteoporotic Fracture Healing

    PubMed Central

    Haffner-Luntzer, Melanie; Kemmler, Julia; Heidler, Verena; Prystaz, Katja; Schinke, Thorsten; Amling, Michael; Kovtun, Anna; Rapp, Anna E.; Ignatius, Anita; Liedert, Astrid

    2016-01-01

    The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications. PMID:27410432

  3. Competitive inhibition of benzodiazepine binding by fractions from porcine brain.

    PubMed Central

    Colello, G D; Hockenbery, D M; Bosmann, H B; Fuchs, S; Folkers, K

    1978-01-01

    Fractions of porcine cerebral cortex extract separated by molecular weight on a Sephadex G-75 column were tested for their activities and potencies to inhibit [3H]benzodiazepine binding to rat brain homogenates. The fractions spanned molecular weights from 500 to 100,000. A potent inhibitor (benzodiazepine-competitive factor I, BCF-I) was discovered in the fraction containing substances with molecular weights from 40,000 to 70,000. Equilibrium binding studies indicated that BCF-I was a competitive inhibitor, making it a candidate as a benzodiazepine endogenous factor or profactor. BCF-I was heat stable, but trypsin digestion destroyed its activity. Another inhibitory fraction (BCF-II) was 1/5th as active as BCF-I and contained substances with molecular weights from 1000 to 2000. Images PMID:32539

  4. Endothelial proteoglycans inhibit bFGF binding and mitogenesis.

    PubMed

    Forsten, K E; Courant, N A; Nugent, M A

    1997-08-01

    Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG-A and PG-B. The large sized fraction (PG-A) had greater inhibitory activity than did PG-B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG-A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG-B. Both proteoglycan fractions contained perlecan-like core proteins; however, PG-A contained an additional core protein (approximately 190 kDa) that was not observed in PG-B. Both proteoglycan fractions bound bFGF directly, and PG-A bound a significantly greater relative amount of bFGF than did PG-B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF-induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis.

  5. Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

    PubMed

    Oh, Y S; Ahn, H S; Gye, M C

    2013-12-01

    Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

  6. Prolyl hydroxylase 3 inhibited the tumorigenecity of gastric cancer cells.

    PubMed

    Cui, Lei; Qu, Jianguo; Dang, Shengchun; Mao, Zhengfa; Wang, Xuqing; Fan, Xin; Sun, Kang; Zhang, Jianxin

    2014-09-01

    Gastric cancer is one of the most common malignancies and the second leading cause of cancer-related death in the world, and it is very urgent to develop novel therapeutic strategies. Although HIF-1α is the most highly characterized target of prolyl hydroxylase 3 (PHD3), PHD3 has been shown to regulate several signal pathways independent of HIF-1α. Here, we found that the expression of PHD3 was decreased in the clinical gastric cancer samples and reversely correlated with tumor size and tumor stage. Over-expression of PHD3 in the gastric cancer cells significantly inhibited cell growth in vitro and in vivo, while knockdown the expression of PHD3 promoted the tumorigenecity of gastric cancer cells. Mechanistically, it showed that PHD3 downregulated the expression of beta-catenin and inhibited beta-catenin/T-cell factor (TCF) signaling. Taken together, our findings demonstrate that PHD3 inhibits gastric cancer by suppressing the beta-catenin/TCF signaling and PHD3 might be an important therapeutic target in gastric cancer.

  7. An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2.

    PubMed

    Rundle, Natalie T; Nelson, Jim; Flory, Mark R; Joseph, Jomon; Th'ng, John; Aebersold, Ruedi; Dasso, Mary; Andersen, Raymond J; Roberge, Michel

    2006-08-22

    Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit the polymerization or depolymerization of tubulin. To identify EKA-interacting proteins, we used a cell-permeable biotinylated form that retains biological activity to isolate binding proteins from living cells. Mass spectrometric analysis identified six EKA-binding proteins, including Ran-binding protein 2, a kinetochore protein whose depletion by small interfering RNA causes a similar mitotic arrest phenotype.

  8. Roles of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression and beta-catenin activation in gastric carcinogenesis in N-methyl-N-nitrosourea-treated K19-C2mE transgenic mice.

    PubMed

    Takasu, Shinji; Tsukamoto, Tetsuya; Cao, Xue-Yuan; Toyoda, Takeshi; Hirata, Akihiro; Ban, Hisayo; Yamamoto, Masami; Sakai, Hiroki; Yanai, Tokuma; Masegi, Toshiaki; Oshima, Masanobu; Tatematsu, Masae

    2008-12-01

    K19-C2mE transgenic (Tg) mice, simultaneously expressing cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in the gastric mucosa under the cytokeratin 19 gene promoter, were here treated with N-methyl-N-nitrosourea (MNU) and inoculated with Helicobacter pylori (H. pylori) to investigate gastric carcinogenesis. Wild-type (WT) and Tg mice undergoing MNU treatment frequently developed tumors in the pyloric region (100% and 94.7%, respectively); multiplicity in Tg was higher than that in WT (P < 0.05) with H. pylori infection. Larger pyloric tumors were more frequently observed in Tg than in WT (P < 0.05). In addition, Tg developed fundic tumors, where WT did not. No gastric tumors were observed without MNU treatment. Transcripts of TNF-alpha, iNOS, IL-1beta, and CXCL14 were up-regulated with H. pylori infection in both genotypes and were also increased more in Tg than in WT within H. pylori-inoculated animals. Immunohistochemical analysis demonstrated significantly greater beta-catenin accumulation in pyloric tumors, compared with those in the fundus (P < 0.01) with mutations of exon 3; 18.2% and 31.6% in MNU-alone and MNU + H. pylori-treated WT, whereas 21.4% and 62.5% was observed in the Tg, respectively; the latter significantly higher (P < 0.05), suggesting the role of H. pylori in Wnt activation. In conclusion, K19-C2mE mice promoted gastric cancer in both fundic and pyloric regions. Furthermore beta-catenin activation may play the important role of pyloric carcinogenesis especially in H. pylori-infected Tg. Induction of various inflammatory cytokines in addition to overexpression of COX-2/mPGES-1 could be risk factors of gastric carcinogenesis and may serve as a better gastric carcinogenesis model.

  9. Green tea catechins inhibit VEGF-induced angiogenesis in vitro through suppression of VE-cadherin phosphorylation and inactivation of Akt molecule.

    PubMed

    Tang, Feng-Yao; Nguyen, Nhan; Meydani, Mohsen

    2003-10-10

    Studies have indicated that the consumption of green tea is associated with a reduced risk of developing certain forms of cancer and angiogenesis. The mechanism of inhibition of angiogenesis by green tea or its catechins, however, has not been well-established. Vascular endothelial (VE)-cadherin, an adhesive molecule located at the site of intercellular contact, is involved in cell-cell recognition during vascular morphogenesis. The extracellular domain of VE-cadherin mediates initial cell adhesion, whereas the cytosolic tail binding with beta-catenin is required for interaction with the cytoskeleton and junctional strength. Therefore, the cadherin-catenin adhesion system is implicated in cell recognition, differentiation, growth and migration of capillary endothelium. Using tube formation of human microvascular endothelial cells (HMVEC) in culture as an in vitro model of angiogenesis, we reported that vascular endothelial growth factor (VEGF)-induced tube formation is inhibited by anti-VE-cadherin antibody and dose-dependently by green tea catechins. We also demonstrated here that inhibition of tube formation by epigallocatechin gallate (EGCG), one of the green tea catechins, is in part mediated through suppression of VE-cadherin tyrosine phosphorylation and inhibition of Akt activation during VEGF-induced tube formation. These findings indicate that VE-cadherin and Akt, known downstream proteins in VEGFR-2-mediated cascade, are the new-targeted proteins by which green tea catechins inhibit angiogenesis.

  10. Inhibition by Elongation Factor EF G of Aminoacyl-tRNA Binding to Ribosomes

    PubMed Central

    Cabrer, Bartolomé; Vázquez, David; Modolell, Juan

    1972-01-01

    Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg++ concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site. PMID:4551985

  11. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  12. Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance.

    PubMed

    Shang, Jing; Piskarev, Vladimir E; Xia, Ming; Huang, Pengwei; Jiang, Xi; Likhosherstov, Leonid M; Novikova, Olga S; Newburg, David S; Ratner, Daniel M

    2013-12-01

    Human milk glycans inhibit binding between norovirus and its host glycan receptor; such competitive inhibition by human milk glycans is associated with a reduced risk of infection. The relationship between the presence of specific structural motifs in the human milk glycan and its ability to inhibit binding by specific norovirus strains requires facile, accurate and miniaturized-binding assays. Toward this end, a high-throughput biosensor platform was developed based on surface plasmon resonance imaging (SPRi) of glycan microarrays. The SPRi was validated, and its utility was tested, by measuring binding specificities between defined human milk glycan epitopes and the capsids of two common norovirus strains, VA387 and Norwalk. Human milk oligosaccharide (HMOS)-based neoglycoconjugates, including chemically derived neoglycoproteins and oligosaccharide-glycine derivatives, were used to represent polyvalent glycoconjugates and monovalent oligosaccharides, respectively, in human milk. SPRi binding results established that the glycan motifs that bind norovirus capsids depend upon strain; VA387 capsid interacts with two neoglycoproteins, whereas Norwalk capsid binds to a different set of HMOS motifs in the form of both polyvalent neoglycoproteins and monovalent oligosaccharides. SPRi competitive binding assays further demonstrated that specific norovirus-binding glycans are able to inhibit norovirus capsid binding to their host receptors. A polyvalent neoglycoconjugate with clustered carbohydrate moieties is required for the inhibition of VA387 capsid binding to host receptor glycans, whereas both monovalent oligosaccharides and polyvalent neoglycoconjugates are able to inhibit Norwalk capsid binding to its host receptor. Binding of HMOS and HMOS-based neoglycoconjugates to norovirus capsids depends upon the specific strain characteristics, implying that HMOS and their polyvalent derivatives are potential anti-adhesive agents for norovirus prophylaxis.

  13. Identifying human milk glycans that inhibit norovirus binding using surface plasmon resonance

    PubMed Central

    Shang, Jing; Piskarev, Vladimir E; Xia, Ming; Huang, Pengwei; Jiang, Xi; Likhosherstov, Leonid M; Novikova, Olga S; Newburg, David S; Ratner, Daniel M

    2013-01-01

    Human milk glycans inhibit binding between norovirus and its host glycan receptor; such competitive inhibition by human milk glycans is associated with a reduced risk of infection. The relationship between the presence of specific structural motifs in the human milk glycan and its ability to inhibit binding by specific norovirus strains requires facile, accurate and miniaturized-binding assays. Toward this end, a high-throughput biosensor platform was developed based on surface plasmon resonance imaging (SPRi) of glycan microarrays. The SPRi was validated, and its utility was tested, by measuring binding specificities between defined human milk glycan epitopes and the capsids of two common norovirus strains, VA387 and Norwalk. Human milk oligosaccharide (HMOS)-based neoglycoconjugates, including chemically derived neoglycoproteins and oligosaccharide-glycine derivatives, were used to represent polyvalent glycoconjugates and monovalent oligosaccharides, respectively, in human milk. SPRi binding results established that the glycan motifs that bind norovirus capsids depend upon strain; VA387 capsid interacts with two neoglycoproteins, whereas Norwalk capsid binds to a different set of HMOS motifs in the form of both polyvalent neoglycoproteins and monovalent oligosaccharides. SPRi competitive binding assays further demonstrated that specific norovirus-binding glycans are able to inhibit norovirus capsid binding to their host receptors. A polyvalent neoglycoconjugate with clustered carbohydrate moieties is required for the inhibition of VA387 capsid binding to host receptor glycans, whereas both monovalent oligosaccharides and polyvalent neoglycoconjugates are able to inhibit Norwalk capsid binding to its host receptor. Binding of HMOS and HMOS-based neoglycoconjugates to norovirus capsids depends upon the specific strain characteristics, implying that HMOS and their polyvalent derivatives are potential anti-adhesive agents for norovirus prophylaxis. PMID:24026239

  14. Downregulation of adenomatous polyposis coli by microRNA-663 promotes odontogenic differentiation through activation of Wnt/beta-catenin signaling

    SciTech Connect

    Kim, Jae-Sung; Park, Min-Gyeong; Lee, Seul Ah; Park, Sun-Young; Kim, Heung-Joong; Yu, Sun-Kyoung; Kim, Chun Sung; Kim, Su-Gwan; Oh, Ji-Su; You, Jae-Seek; Kim, Jin-Soo; Seo, Yo-Seob; Chun, Hong Sung; Park, Joo-Cheol; Kim, Do Kyung

    2014-04-18

    Highlights: • miR-663 is significantly up-regulated during MDPC-23 odontoblastic cell differentiation. • miR-663 accelerates mineralization in MDPC-23 odontoblastic cells without cell proliferation. • miR-663 promotes odontoblastic cell differentiation by targeting APC and activating Wnt/β-catenin signaling in MDPC-23 cells. - Abstract: MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/β-catenin signaling pathway, has a miR-663 binding site in its 3′-untranslated region (3′UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/β-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.

  15. Psoralens potentiate ultraviolet light-induced inhibition of epidermal growth factor binding

    SciTech Connect

    Laskin, J.D.; Lee, E.; Laskin, D.L.; Gallo, M.A.

    1986-11-01

    The psoralens, when activated by ultraviolet light of 320-400 nm (UVA light), are potent modulators of epidermal cell growth and differentiation. Previously, we reported that, in mammalian cells, these compounds bind to specific saturable high-affinity cellular receptor sites. In the present studies, we demonstrate that binding of psoralens to their receptors followed by UVA light activation is associated with inhibition of epidermal growth factor (EGF) receptor binding. Inhibition of EGF binding, which required UVA light, was rapid and dependent on the dose of UVA light (0.5-2.0 J/cm2), as well as the concentration of psoralens (10 nM to 1 microM). Higher doses of UVA light (2.0-6.0 J/cm2) by themselves were also inhibitory, indicating that psoralens potentiate the UVA-induced inhibition of EGF binding. A number of biologically active analogs of psoralen, including 8-methoxypsoralen, 5-methoxypsoralen, and 4,5',8-trimethylpsoralen, when activated by UVA light, were found to be inhibitors of binding. Inhibition of EGF binding by psoralens was observed in a variety of human and mouse cell culture lines known to possess psoralen receptors. In the epidermal-derived line PAM 212, at least two populations of receptors with different affinities for EGF were found. Psoralens and UVA light selectively inhibited binding to the higher-affinity EGF receptors, an effect analogous to that of the phorbol ester tumor promoters. As observed with phorbol esters, photoactivated psoralens appeared to inhibit EGF binding by an indirect mechanism. These data demonstrate that the psoralens and UVA light have direct biological effects on cell-surface membranes. Since EGF is a growth-regulatory peptide, the ability of psoralens and UVA light to inhibit EGF binding may underlie the biologic effects of these agents in the skin.

  16. Water may inhibit oxygen binding in hemoprotein models.

    PubMed

    Collman, James P; Decréau, Richard A; Dey, Abhishek; Yang, Ying

    2009-03-17

    Three distal imidazole pickets in a cytochrome c oxidase (CcO) model form a pocket hosting a cluster of water molecules. The cluster makes the ferrous heme low spin, and consequently the O(2) binding slow. The nature of the rigid proximal imidazole tail favors a high spin/low spin cross-over. The O(2) binding rate is enhanced either by removing the water, increasing the hydrophobicity of the gas binding pocket, or inserting a metal ion that coordinates to the 3 distal imidazole pickets.

  17. Sticky Plans: Inhibition and Binding during Serial-Task Control

    ERIC Educational Resources Information Center

    Mayr, Ulrich

    2009-01-01

    Recent evidence suggests substantial response-time costs associated with lag-2 repetitions of tasks within explicitly controlled task sequences [Koch, I., Philipp, A. M., Gade, M. (2006). Chunking in task sequences modulates task inhibition. "Psychological Science," 17, 346-350; Schneider, D. W. (2007). Task-set inhibition in chunked task…

  18. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

    PubMed Central

    Miller, S D; Farmer, G; Prives, C

    1995-01-01

    The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition. PMID:8524220

  19. Competitive inhibition of (TH)dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    SciTech Connect

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-03-01

    The inhibitory effect of leupeptin on (TH)dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of (TH)dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of (TH)dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

  20. Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy.

    PubMed

    Yang, Yong-Jun; Liu, Wei-Min; Zhou, Jia-Xi; Cao, Yu-Jing; Li, Jing; Peng, Sha; Wang, Li; Yuan, Jiang-Gang; Duan, En-Kui

    2006-01-11

    Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.

  1. Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase.

    PubMed

    Cardinale, Daniela; Guaitoli, Giambattista; Tondi, Donatella; Luciani, Rosaria; Henrich, Stefan; Salo-Ahen, Outi M H; Ferrari, Stefania; Marverti, Gaetano; Guerrieri, Davide; Ligabue, Alessio; Frassineti, Chiara; Pozzi, Cecilia; Mangani, Stefano; Fessas, Dimitrios; Guerrini, Remo; Ponterini, Glauco; Wade, Rebecca C; Costi, M Paola

    2011-08-23

    Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

  2. Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding

    PubMed Central

    Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

    2016-01-01

    Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3. PMID:27458147

  3. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    PubMed

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  4. Rapid kinetics of 2-adrenergic agonist binding and inhibition of adenylate cyclase

    SciTech Connect

    Thomsen, W.; Neubig, R.R.

    1987-05-01

    Activation of 2-adrenergic receptors in human platelets results in inhibition of adenylate cyclase (AC). To elucidate the relation between agonist binding and response, the authors have used a novel rapid-mix quench method to compare the kinetics of binding and response. At functionally effective concentrations, the time course of binding of the full 2-agonist, (TH)UK14,304 (UK), to purified platelet membranes was faster than could be measured manually. Using the rapid-mix quench method, agonist binding was quantitated for times for 0.3 to 60 seconds. UK binding exhibited biexponential kinetics. The rate constant of the fast binding component increases linearly with agonist concentration from 1 to 100 nM with a second order rate constant and 7 x 10WM s (at 25C). The slow rate constant was nearly independent of agonist concentration. The half times of the fast and slow components of binding for 100 nM UK are 1.5 seconds and approximately 2 minutes respectively. The rate and magnitude of the fast binding was unaffected by 10 M GTP whereas the magnitude of the slow phase was markedly reduced. Inhibition of forskolin stimulated AC by 100 M epinephrine occurs with a lag of 5-10 seconds in the presence of 10 M GTP. At lower GTP concentrations, this lag is prolonged. The observation that the fast component of agonist binding precedes inhibition even at agonist concentrations 20-fold lower than the EC40 for responses indicates that the rate limiting step in inhibition of AC is distal to the binding of agonist.

  5. Hydroxyapatite-binding peptides for bone growth and inhibition

    DOEpatents

    Bertozzi, Carolyn R.; Song, Jie; Lee, Seung-Wuk

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  6. Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

    PubMed

    Baureithel, K H; Büter, K B; Engesser, A; Burkard, W; Schaffner, W

    1997-06-01

    Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

  7. Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul

    PubMed Central

    2013-01-01

    Background z-Guggulsterone (z-Gug) and Gugulipid (GL) have been used to treat a variety of ailments. We now report their anti-cancer effect and mechanism against human breast cancer. Methods Using the human estrogen receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cells as well as the normal human mammary epithelial cell line (HMEC), we evaluated the anti-breast-cancer efficacy and apoptosis inducing activity of GL. We determined the cellular and molecular mechanism of GL-inhibited breast cancer cell growth. Results GL significantly inhibited growth of MCF-7 and MDA-MB-231 cells with an IC50~2 μM at pharmacologically relevant concentrations standardized to its major active constituent z-Gug. The GL-induced growth inhibition correlated with apoptosis induction as evidenced by an increase in cytoplasmic histone-associated DNA fragmentation and caspase 3 activity. The GL-induced apoptosis was associated with down-regulation of the β-Catenin signaling pathway. The decreased expression of Wnt/β-Catenin targeting genes, such as cyclin D1, C-myc and survivin, and the inhibition of the activity of the transcription factor (T-cell factor 4, TCF-4) were observed in GL-treated breast cancer cells. The GL treatment resulted in a significant reduction of β-Catenin /TCF-4 complex in both of the cancer cells. The GL-induced apoptotic cell death was significantly enhanced by RNA Interference of β-Catenin and TCF-4. On the other hand, the normal human mammary epithelial cell HMEC, compared with the human breast cancer cells, is significantly more resistant to growth inhibition and apoptosis induction by GL. Conclusion The present study indicates that the β-Catenin signaling pathway is the target for GL-induced growth inhibition and apoptosis in human breast cancer. PMID:23914993

  8. Computational insights into the suicide inhibition of Plasmodium falciparum Fk506-binding protein 35.

    PubMed

    MacDonald, Corey A; Boyd, Russell J

    2015-08-15

    Malaria is a parasite affecting millions of people worldwide. With the risk of malarial resistance reaching catastrophic levels, novel methods into the inhibition of this disease need to be prioritized. The exploitation of active site differences between parasitic and human peptidyl-prolyl cis/trans isomerases can be used for suicide inhibition, effectively poisoning the parasite without affecting the patient. This method of inhibition was explored using Plasmodium falciparum and Homo sapiens Fk506-binding proteins as templates for quantum mechanics/molecular mechanics calculations. Modification of the natural substrate has shown suicide inhibition is a valid approach for novel anti-malarials with little risk for parasitic resistance.

  9. A nucleolar localizing Rev binding element inhibits HIV replication

    PubMed Central

    Michienzi, Alessandro; De Angelis, Fernanda G; Bozzoni, Irene; Rossi, John J

    2006-01-01

    The Rev protein of the human immunodeficiency virus (HIV) facilitates the nuclear export of intron containing viral mRNAs allowing formation of infectious virions. Rev traffics through the nucleolus and shuttles between the nucleus and cytoplasm. Rev multimerization and interaction with the export protein CRM1 takes place in the nucleolus. To test the importance of Rev nucleolar trafficking in the HIV-1 replication cycle, we created a nucleolar localizing Rev Response Element (RRE) decoy and tested this for its anti-HIV activity. The RRE decoy provided marked inhibition of HIV-1 replication in both the CEM T-cell line and in primary CD34+ derived monocytes. These results demonstrate that titration of Rev in the nucleolus impairs HIV-1 replication and supports a functional role for Rev trafficking in this sub-cellular compartment. PMID:16712721

  10. Pharmacological inhibition of mannose-binding lectin ameliorates neurobehavioral dysfunction following experimental traumatic brain injury.

    PubMed

    De Blasio, Daiana; Fumagalli, Stefano; Longhi, Luca; Orsini, Franca; Palmioli, Alessandro; Stravalaci, Matteo; Vegliante, Gloria; Zanier, Elisa R; Bernardi, Anna; Gobbi, Marco; De Simoni, Maria-Grazia

    2017-03-01

    Mannose-binding lectin is present in the contusion area of traumatic brain-injured patients and in that of traumatic brain-injured mice, where mannose-binding lectin-C exceeds mannose-binding lectin-A. The reduced susceptibility to traumatic brain injury of mannose-binding lectin double knock-out mice (mannose-binding lectin(-/-)) when compared to wild type mice suggests that mannose-binding lectin may be a therapeutic target following traumatic brain injury. Here, we evaluated the effects of a multivalent glycomimetic mannose-binding lectin ligand, Polyman9, following traumatic brain injury in mice. In vitro surface plasmon resonance assay indicated that Polyman9 dose-dependently inhibits the binding to immobilized mannose residues of plasma mannose-binding lectin-C selectively over that of mannose-binding lectin-A. Male C57Bl/6 mice underwent sham/controlled cortical impact traumatic brain injury and intravenous treatment with Polyman9/saline. Ex-vivo surface plasmon resonance studies confirmed that Polyman9 effectively reduces the binding of plasma mannose-binding lectin-C to immobilized mannose residues. In vivo studies up to four weeks post injury, showed that Polyman9 induces significant improvement in sensorimotor deficits (by neuroscore and beam walk), promotes neurogenesis (73% increase in doublecortin immunoreactivity), and astrogliosis (28% increase in glial fibrillary acid protein). Polyman9 administration in brain-injured mannose-binding lectin(-/-) mice had no effect on post-traumatic brain-injured functional deficits, suggestive of the specificity of its neuroprotective effects. The neurobehavioral efficacy of Polyman9 implicates mannose-binding lectin-C as a novel therapeutic target for traumatic brain injury.

  11. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/beta-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans.

    PubMed

    Mink, M; Fogelgren, B; Olszewski, K; Maroy, P; Csiszar, K

    2001-06-01

    A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.

  12. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

    SciTech Connect

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  13. Phytochrome B inhibits binding of Phytochrome-Interacting Factors to their target promoters

    PubMed Central

    Park, Eunae; Park, Jeongmoo; Kim, Junghyun; Nagatani, Akira; Lagarias, J. Clark; Choi, Giltsu

    2012-01-01

    Summary Phytochromes are red and far-red light receptors in plants that mediate critical responses to light throughout the life cycle. They achieve this in part by targeting negatively acting bHLH transcription factors called phytochrome-interacting factors (PIFs) for degradation within the nucleus. It is not known, however, if protein degradation is the primary mechanism by which phytochromes inhibit these repressors of photomorphogenesis. Here, we use ChIP analysis to show that phyB inhibits the regulatory activity of PIF1 and PIF3 by releasing them from their DNA targets. The N-terminal fragment of phyB (NG-GUS-NLS; NGB) also inhibits the binding of PIF3 to its target promoters. Unlike the full-length phyB, however, NGB does not promote PIF3 degradation, establishing the activity of NGB reflects its ability to inhibit PIFs’ binding to DNA. We further show that Pfr forms of both full-length phyB and NGB inhibit the DNA binding of PIF1 and PIF3 in vitro. Taken together, our results indicate that phyB inhibition of PIF function involves two separate processes, sequestration and protein degradation. PMID:22849408

  14. TIMP-2 modulates cancer cell transcriptional profile and enhances E-cadherin/beta-catenin complex expression in A549 lung cancer cells

    PubMed Central

    Bourboulia, Dimitra; Han, HuiYing; Isaac, Biju; Wei, Beiyang; Neckers, Len; Stetler-Stevenson, William G.

    2013-01-01

    Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) plays an essential role in regulating matrix remodeling, cell growth, differentiation, angiogenesis and apoptosis in vitro and in vivo. We have recently shown that TIMP-2-mediated inhibition of tumor growth is independent of matrix metalloproteinase-mediated mechanisms, and is a consequence of modulating both the tumor cells and the tumor microenvironment. In the current study we aim to identify the molecular pathways associated with these effects. We analyzed the transcriptional profile of the human lung cancer cell line A549 upon overexpression of TIMP-2 and Ala+TIMP-2 (mutant that does not inhibit MMP activity), and we found changes in gene expression predominantly related to decreased tumor development and metastasis. Increased E-cadherin expression in response to both TIMP-2 and Ala+TIMP-2 expression was confirmed by real time quantitative RT-PCR and immunoblotting. A549 cells treated with epidermal growth factor (EGF) displayed loss of cobblestone morphology and cell-cell contact, while cells overexpressing TIMP-2 or Ala+TIMP-2 were resistant to EGF-induced morphological changes. Moreover, exogenous treatment with recombinant Ala+TIMP-2 blocked EGF induced down-regulation of E-cadherin. In vivo, immunohistochemistry of A549 xenografts expressing either TIMP-2 or Ala+TIMP-2 demonstrated increased E-cadherin protein levels. More importantly, transcriptional profile analysis of tumor tissue revealed critical pathways associated with effects on tumor-host interaction and inhibition of tumor growth. In conclusion, we show that TIMP-2 promotes an anti-tumoral transcriptional profile in vitro and in vivo, including upregulation of E-cadherin, in A549 lung cancer cells. PMID:23371049

  15. A Common Anesthetic Binding Site for Inhibition of Pentameric Ligand-gated Ion Channels

    PubMed Central

    Kinde, Monica N.; Bu, Weiming; Chen, Qiang; Xu, Yan; Eckenhoff, Roderic G.; Tang, Pei

    2016-01-01

    Background Identifying functionally relevant anesthetic binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC ELIC, but the sites responsible for functional inhibition remain undetermined. Methods We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed 19F NMR to support propofol binding to a transmembrane domain (TMD) intra-subunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-GABAAR chimera that replaced the ELIC TMD with the α1β3GABAAR TMD and compared functional responses of ELIC-GABAAR and ELIC to propofol modulations. Results Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD), but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intra-subunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. 19F NMR supported propofol binding to this TMD intra-subunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. Conclusions The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intra-subunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs. PMID:26756520

  16. Binding modes of decavanadate to myosin and inhibition of the actomyosin ATPase activity.

    PubMed

    Tiago, Teresa; Martel, Paulo; Gutiérrez-Merino, Carlos; Aureliano, Manuel

    2007-04-01

    Decavanadate, a vanadate oligomer, is known to interact with myosin and to inhibit the ATPase activity, but the putative binding sites and the mechanism of inhibition are still to be clarified. We have previously proposed that the decavanadate (V(10)O(28)(6-)) inhibition of the actin-stimulated myosin ATPase activity is non-competitive towards both actin and ATP. A likely explanation for these results is that V(10) binds to the so-called back-door at the end of the Pi-tube opposite to the nucleotide-binding site. In order to further investigate this possibility, we have carried out molecular docking simulations of the V(10) oligomer on three different structures of the myosin motor domain of Dictyostelium discoideum, representing distinct states of the ATPase cycle. The results indicate a clear preference of V(10) to bind at the back-door, but only on the "open" structures where there is access to the phosphate binding-loop. It is suggested that V(10) acts as a "back-door stop" blocking the closure of the 50-kDa cleft necessary to carry out ATP-gamma-phosphate hydrolysis. This provides a simple explanation to the non-competitive behavior of V(10) and spurs the use of the oligomer as a tool to elucidate myosin back-door conformational changes in the process of muscle contraction.

  17. Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells.

    PubMed

    Weng, Tsui-Ying; Chen, Lien-Cheng; Shyu, Huey-Wen; Chen, Shun-Hua; Wang, Jen-Ren; Yu, Chun-Keung; Lei, Huan-Yao; Yeh, Trai-Ming

    2005-07-01

    The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-alpha expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.

  18. Wnt/beta-catenin signaling down-regulates N-acetylglucosaminyltransferase III expression: the implications of two mutually exclusive pathways for regulation.

    PubMed

    Xu, Qingsong; Akama, Ryota; Isaji, Tomoya; Lu, Yingying; Hashimoto, Hirokazu; Kariya, Yoshinobu; Fukuda, Tomohiko; Du, Yuguang; Gu, Jianguo

    2011-02-11

    In previous studies, we reported that N-acetylglucosaminyltransferase III (GnT-III) activity and the enzyme product, bisected N-glycans, both were induced in cells cultured under dense conditions in an E-cadherin-dependent manner (Iijima, J., Zhao, Y., Isaji, T., Kameyama, A., Nakaya, S., Wang, X., Ihara, H., Cheng, X., Nakagawa, T., Miyoshi, E., Kondo, A., Narimatsu, H., Taniguchi, N., and Gu, J. (2006) J. Biol. Chem. 281, 13038-13046). Furthermore, we found that α-catenin, a component of the E-cadherin-catenin complex, was also required for this induction (Akama, R., Sato, Y., Kariya, Y., Isaji, T., Fukuda, T., Lu, L., Taniguchi, N., Ozawa, M., and Gu, J. (2008) Proteomics 8, 3221-3228). To further explore the molecular mechanism of this regulation, the roles of β-catenin, an essential molecule in both cadherin-mediated cell adhesion and canonical Wnt signaling, were investigated. Unexpectedly, shRNA knockdown of β-catenin resulted in a dramatic increase in GnT-III expression and its product, the bisected N-glycans, which was confirmed by RT-PCR and GnT-III activity and by E4-PHA lectin blot analysis. The induction of GnT-III expression increased bisecting GlcNAc residues on β1 integrin, which led to down-regulation of integrin-mediated cell adhesion and cell migration. Immunostaining showed that nuclear localization of β-catenin was greatly suppressed; intriguingly, the knockdown of β-catenin in the nuclei was more effective than that in cell-cell contacts in the knockdown cells, which was also confirmed by Western blot analysis. Stimulation of the Wnt signaling pathway by the addition of exogenous Wnt3a or BIO, a GSK-3β inhibitor, consistently and significantly inhibited GnT-III expression and its products. Conversely, the inhibition of β-catenin translocation into the nuclei increased GnT-III activation. Taken together, the results of the present study are the first to clearly demonstrate that GnT-III expression may be precisely regulated by the

  19. Tumor suppressor berberine binds VASP to inhibit cell migration in basal-like breast cancer

    PubMed Central

    Wang, Xiaolan; Kuang, Changchun; Xiang, Qingmin; Yang, Fang; Xiang, Jin; Zhu, Shan; Wei, Lei; Zhang, Jingwei

    2016-01-01

    Berberine is a plant-derived compound used in traditional Chinese medicine, which has been shown to inhibit cell proliferation and migration in breast cancer. On the other hand, vasodilator-stimulated phosphoprotein (VASP) promotes actin filament elongation and cell migration. We previously showed that VASP is overexpressed in high-motility breast cancer cells. Here we investigated whether the anti-tumorigenic effects of berberine are mediated by binding VASP in basal-like breast cancer. Our results show that berberine suppresses proliferation and migration of MDA-MB-231 cells as well as tumor growth in MDA-MB-231 nude mouse xenografts. We also show that berberine binds to VASP, inducing changes in its secondary structure and inhibits actin polymerization. Our study reveals the mechanism underlying berberine's inhibition of cell proliferation and migration in basal-like breast cancer, highlighting the use of berberine as a potential adjuvant therapeutic agent. PMID:27322681

  20. Chronic chemotherapeutic stress promotes evolution of stemness and WNT/beta-catenin signaling in colorectal cancer cells: implications for clinical use of WNT-signaling inhibitors

    PubMed Central

    Ayadi, Meriam; Bouygues, Anaïs; Ouaret, Djamila; Ferrand, Nathalie; Chouaib, Salem; Thiery, Jean-Paul; Muchardt, Christian; Sabbah, Michèle; Larsen, Annette K

    2015-01-01

    Most solid tumors contain a subfraction of cells with stem/progenitor cell features. Stem cells are naturally chemoresistant suggesting that chronic chemotherapeutic stress may select for cells with increased “stemness”. We carried out a comprehensive molecular and functional analysis of six independently selected colorectal cancer (CRC) cell lines with acquired resistance to three different chemotherapeutic agents derived from two distinct parental cell lines. Chronic drug exposure resulted in complex alterations of stem cell markers that could be classified into three categories: 1) one cell line, HT-29/5-FU, showed increased “stemness” and WNT-signaling, 2) three cell lines showed decreased expression of stem cell markers, decreased aldehyde dehydrogenase activity, attenuated WNT-signaling and lost the capacity to form colonospheres and 3) two cell lines displayed prominent expression of ABC transporters with a heterogeneous response for stem cell markers. While WNT-signaling could be attenuated in the HT-29/5-FU cells by the WNT-signaling inhibitors ICG-001 and PKF-118, this was not accompanied by any selective growth inhibitory effect suggesting that the cytotoxic activity of these compounds is not directly linked to WNT-signaling inhibition. We conclude that classical WNT-signaling inhibitors have toxic off-target activities that need to be addressed for clinical development. PMID:26041882

  1. Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts.

    PubMed

    Breiner, M; Romalo, G; Schweikert, H U

    1986-08-15

    The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 degrees C with 2 nM 3H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (Ki) were determined as the concentration of competitor-required for 50% inhibition of 3H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of 3H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone. Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have Ki values similar to testosterone. An order of magnitude lower Ki values (around 10(-7) M) were found for the androgen 17 alpha-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) greater than glucocorticoids as well as aromatase inhibitors and potassium canrenoate.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Inhibition of tobacco mosaic virus movement by expression of an actin-binding protein.

    PubMed

    Hofmann, Christina; Niehl, Annette; Sambade, Adrian; Steinmetz, André; Heinlein, Manfred

    2009-04-01

    The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

  3. Binding of dihydroxynaphthyl aryl ketones to tubulin colchicine site inhibits microtubule assembly.

    PubMed

    Gutierrez, Eunices; Benites, Julio; Valderrama, Jaime A; Calderon, Pedro Buc; Verrax, Julien; Nova, Esteban; Villanelo, Felipe; Maturana, Daniel; Escobar, Cristian; Lagos, Rosalba; Monasterio, Octavio

    2015-10-23

    Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 μM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 μM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

  4. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation.

    PubMed

    Klatt, P; Molina, E P; Lamas, S

    1999-05-28

    This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.

  5. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

    PubMed Central

    Chadee, K; Petri, W A; Innes, D J; Ravdin, J I

    1987-01-01

    Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells. Images PMID:2890655

  6. Inhibition of pulmonary fibrosis in mice by CXCL10 requires glycosaminoglycan binding and syndecan-4

    PubMed Central

    Jiang, Dianhua; Liang, Jiurong; Campanella, Gabriele S.; Guo, Rishu; Yu, Shuang; Xie, Ting; Liu, Ningshan; Jung, Yoosun; Homer, Robert; Meltzer, Eric B.; Li, Yuejuan; Tager, Andrew M.; Goetinck, Paul F.; Luster, Andrew D.; Noble, Paul W.

    2010-01-01

    Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4–null (Sdc4–/–) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4–/– mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis. PMID:20484822

  7. Inhibition of NF-kappaB DNA binding by nitric oxide.

    PubMed Central

    Matthews, J R; Botting, C H; Panico, M; Morris, H R; Hay, R T

    1996-01-01

    It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes. PMID:8710491

  8. Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1) by direct protein binding

    SciTech Connect

    Naidu, M.; Naidu, M.; Agarwal, R.; Pena, L.A.; Cunha, L.; Mezei, M.; Shen, M.; Wilson, D.M.; Liu, Y.; Sanchez, Z.; Chaudhary, P.; Wilson, S.H.; Waring, M.J.

    2011-09-15

    Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC{sub 50} values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 {mu}M and 80 nM, respectively. The K{sub D} values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e

  9. Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

    SciTech Connect

    Chauhan, A.; Cauhan, V.P.S.; Deshmukh, D.S.; Brokerhoff, H. )

    1989-06-13

    Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}), can also activate PKC in the presence of phosphatidylserine (PS) and Ca{sup 2+} with a K{sub PIP{sub 2}} of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP{sub 2} and DG on PKC. Here, the authors investigate the effect of PIP{sub 2} on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP{sub 2} inhibited specific binding of ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP{sub 2} than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP{sub 2} is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (K{sub d{prime}}) against PIP{sub 2} concentration was linear over a range of 0.01-1 mol % with a K{sub i} of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP{sub 2}. Competition between PIP{sub 2} and phorbol ester could be determined in a liposomal assay system also. These results indicate that PIP{sub 2}, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP{sub 2} is a primary activator of the enzyme.

  10. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.

  11. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    NASA Astrophysics Data System (ADS)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  12. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    PubMed Central

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches. PMID:26558346

  13. HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

    PubMed

    Mercenne, Gaëlle; Bernacchi, Serena; Richer, Delphine; Bec, Guillaume; Henriet, Simon; Paillart, Jean-Christophe; Marquet, Roland

    2010-01-01

    The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

  14. HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

    PubMed Central

    Mercenne, Gaëlle; Bernacchi, Serena; Richer, Delphine; Bec, Guillaume; Henriet, Simon; Paillart, Jean-Christophe; Marquet, Roland

    2010-01-01

    The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3′UTR than for the 5′UTR, even though this region contained at least one high affinity Vif binding site (apparent Kd = 27 ± 6 nM). Several Vif binding sites were identified in 5′ and 3′UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5′UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes. PMID:19910370

  15. Inhibition of brain [(3)H]cimetidine binding by improgan-like antinociceptive drugs.

    PubMed

    Stadel, Rebecca; Carpenter, Amanda B; Nalwalk, Julia W; de Esch, Iwan J P; Janssen, Elwin; Hough, Lindsay B

    2010-04-25

    [(3)H]cimetidine, a radiolabeled histamine H(2) receptor antagonist, binds with high affinity to an unknown hemoprotein in the brain which is not the histamine H(2) receptor. Improgan, a close chemical congener of cimetidine, is a highly effective pain-relieving drug following CNS administration, yet its mechanism of action remains unknown. To test the hypothesis that the [(3)H]cimetidine-binding site is the improgan antinociceptive target, improgan, cimetidine, and 8 other chemical congeners were studied as potential inhibitors of [(3)H]cimetidine binding in membrane fractions from the rat brain. All compounds produced a concentration-dependent inhibition of [(3)H]cimetidine binding over a 500-fold range of potencies (K(i) values were 14.5 to >8000nM). However, antinociceptive potencies in rats did not significantly correlate with [(3)H]cimetidine-binding affinities (r=0.018, p=0.97, n=10). These results suggest that the [(3)H]cimetidine-binding site is not the analgesic target for improgan-like drugs.

  16. Curcumin inhibits Zika and chikungunya virus infection by inhibiting cell binding.

    PubMed

    Mounce, Bryan C; Cesaro, Teresa; Carrau, Lucia; Vallet, Thomas; Vignuzzi, Marco

    2017-03-24

    Several compounds extracted from spices and herbs exhibit antiviral effects in vitro, suggesting potential pharmacological uses. Curcumin, a component of turmeric, has been used as a food additive and herbal supplement due to its potential medicinal properties. Previously, curcumin exhibited antiviral properties against several viruses, including dengue virus and hepatitis C virus, among others. Here, we describe the antiviral effect of curcumin on Zika and chikungunya viruses, two mosquito-borne outbreak viruses. Both viruses responded to treatment of cells with up to 5 μM curumin without impacting cellular viability. We observed that direct treatment of virus with curcumin reduced infectivity of virus in a dose- and time-dependent manner for these enveloped viruses, as well as vesicular stomatitis virus. In contrast, we found no change in infectivity for Coxsackievirus B3, a non-enveloped virus. Derivatives of curcumin also exhibited antiviral activity against enveloped viruses. Further examination revealed that curcumin interfered with the binding of the enveloped viruses to cells in a dose-dependent manner, though the integrity of the viral RNA was maintained. Together, these results expand the family of viruses sensitive to curcumin and provide a mechanism of action for curcumin's effect on these enveloped viruses.

  17. Cellular Binding of Anionic Nanoparticles is Inhibited by Serum Proteins Independent of Nanoparticle Composition.

    PubMed

    Fleischer, Candace C; Kumar, Umesh; Payne, Christine K

    2013-09-01

    Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

  18. A Conjugate of Two tPA-Binding RNA Aptamers Efficiently Inhibits Fibrinolysis.

    PubMed

    Bjerregaard, Nils; Dupont, Daniel M; Andreasen, Peter A

    2017-04-01

    Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.

  19. Activin inhibits binding of transcription factor Pit-1 to the growth hormone promoter.

    PubMed Central

    Struthers, R S; Gaddy-Kurten, D; Vale, W W

    1992-01-01

    Activin A is a potent growth and differentiation factor related to transforming growth factor beta. In somatotrophs, activin suppresses the biosynthesis and secretion of growth hormone (GH) and cellular proliferation. We report here that, in MtTW15 somatotrophic tumor cells, activin decreased GH mRNA levels and inhibited expression of transfected GH promoter--chloramphenicol acetyltransferase fusion genes. Deletion mapping of nucleotide sequences mediating this inhibition led to the identification of a region that has previously been characterized as binding the pituitary-specific transcription factor Pit-1/GHF-1. Characterization of nuclear factor binding to this region demonstrated that binding of Pit-1 to the GH promoter is lost on activin treatment. These results indicate that activin-induced repression of GH biosynthesis is mediated by the loss of tissue-specific transcription factor binding to the GH promoter and suggest a possible general mechanism for other activin responses, whereby activin regulates the function of other POU- or homeodomain-containing transcription factors. Images PMID:1454833

  20. Beta Catenin in Prostate Cancer Apoptosis

    DTIC Science & Technology

    2014-04-01

    insensitive cell types to determine whether this apoptosis pathway is only specific to androgen sensitive cell types. In order to determine the role of β...obtained: Effect of TRAIL-TZD combination on the apoptosis potential and β-catenin expression of androgen sensitive and androgen insensitive prostate...and 22RV1) and androgen insensitive (DU145 and PC3) prostate cancer cells were treated with either DMSO or a combination of 100ng/ml TRAIL and

  1. Beta Catenin in Prostate Cancer Apoptosis

    DTIC Science & Technology

    2013-04-01

    to be injected with LNCaP prostate cancer cells and following approval from Loyola IACUC and ACURO, the xenograft studies were initiated. As...xenograft studies. 12. Obtained Loyola -IACUC and ACURO approval to utilize the C4-2 and C4-2B cells for in vivo studies. Reportable Outcomes: We had

  2. A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

    PubMed

    Suarez, J E; Urquiza, M; Curtidor, H; Rodriguez, L E; Ocampo, M; Torres, E; Guzman, F; Patarroyo, M E

    2000-01-01

    The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

  3. Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

    PubMed

    Brown, A C; Koufos, E; Balashova, N V; Boesze-Battaglia, K; Lally, E T

    2016-02-01

    The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

  4. Inhibition of LtxA Toxicity by Blocking Cholesterol Binding With Peptides

    PubMed Central

    Brown, Angela C.; Koufos, Evan; Balashova, Nataliya; Boesze-Battaglia, Kathleen; Lally, Edward T.

    2015-01-01

    Summary The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1 (LFA-1), a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of 334LEEYSKR340, in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC336 motif of LtxA (CRAC336WT). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of RTX-producing organisms. PMID:26352738

  5. Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

    PubMed Central

    Bailey, Megan E.; Sackett, Dan L.; Ross, Jennifer L.

    2015-01-01

    Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin. PMID:26682813

  6. 3-Hydroxypyridin-2-thione as Novel Zinc Binding Group for Selective Histone Deacetylase Inhibition

    PubMed Central

    Patil, Vishal; Sodji, Quaovi H.; Kornacki, James R.; Mrksich, Milan; Oyelere, Adegboyega K.

    2013-01-01

    Small molecules bearing hydroxamic acid as the zinc binding group (ZBG) have been the most effective histone deacetylase inhibitor (HDACi) to date. However, concerns about the pharmacokinetic liabilities of the hydroxamic acid moiety have stimulated research efforts aimed at finding alternative non-hydroxamate ZBGs. We have identified 3-hydroxypyridin-2-thione (3-HPT) as a novel ZBG that is compatible with HDAC inhibition. 3-HPT inhibits HDAC 6 and HDAC 8 with an IC50 of 681 nM and 3675 nM respectively. Remarkably, 3-HPT gives no inhibition of HDAC 1. Subsequent optimization led to several novel 3HPT-based HDACi that are selective for HDAC 6 and HDAC 8. Furthermore, a subset of these inhibitors induces apoptosis in various cancer cell lines. PMID:23547652

  7. 3-Hydroxypyridin-2-thione as novel zinc binding group for selective histone deacetylase inhibition.

    PubMed

    Patil, Vishal; Sodji, Quaovi H; Kornacki, James R; Mrksich, Milan; Oyelere, Adegboyega K

    2013-05-09

    Small molecules bearing hydroxamic acid as the zinc binding group (ZBG) have been the most effective histone deacetylase inhibitors (HDACi) to date. However, concerns about the pharmacokinetic liabilities of the hydroxamic acid moiety have stimulated research efforts aimed at finding alternative nonhydroxamate ZBGs. We have identified 3-hydroxypyridin-2-thione (3-HPT) as a novel ZBG that is compatible with HDAC inhibition. 3-HPT inhibits HDAC 6 and HDAC 8 with an IC50 of 681 and 3675 nM, respectively. Remarkably, 3-HPT gives no inhibition of HDAC 1. Subsequent optimization led to several novel 3HPT-based HDACi that are selective for HDAC 6 and HDAC 8. Furthermore, a subset of these inhibitors induces apoptosis in various cancer cell lines.

  8. Thiol-modifying phenylarsine oxide inhibits guanine nucleotide binding of Rho but not of Rac GTPases.

    PubMed

    Gerhard, Ralf; John, Harald; Aktories, Klaus; Just, Ingo

    2003-06-01

    Phenylarsine oxide (PAO) is a phosphotyrosine phosphatase inhibitor that cross-links vicinal thiol groups, thereby inactivating phosphatases possessing XCysXXCysX motifs. The RhoA-GTPase, but not the Rac1-GTPase, also possesses vicinal cysteines within the guanine nucleotide-binding region (aa 13-20) and the phosphohydrolase activity site. Treatment of Caco-2 cells with PAO showed a dose-dependent reorganization of the actin cytoskeleton, indicating involvement of Rho GTPases. As tested by pull-down experiments, RhoA, but not Rac1, from cell lysates was inactivated by PAO in a concentration-dependent manner. Modification of RhoA by PAO resulted in altered mobility on SDS-polyacrylamide gel electrophoresis, and PAO-modified RhoA was no longer substrate for C3-catalyzed ADP-ribosylation. Furthermore, RhoA treated with PAO, but not Rac1 treated with PAO, lost its property to bind to guanine nucleotides. Matrix-assisted laser desorption ionization-mass analysis of PAO-modified RhoA showed a mass shift according to an adduction of a single PAO molecule per molecule RhoA. Further analysis of Glu-C-generated RhoA peptides confirmed binding of PAO to a peptide harboring the guanine nucleotide binding region. Thus, PAO does not exclusively inhibit phosphotyrosine phosphatases but also inactivates RhoA by alteration of nucleotide binding.

  9. Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides.

    PubMed

    Liron, Zvi; Tender, Leonard M; Golden, Joel P; Ligler, Frances S

    2002-06-01

    Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.

  10. Identification of an allosteric binding site for RORγt inhibition

    PubMed Central

    Scheepstra, Marcel; Leysen, Seppe; van Almen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors. PMID:26640126

  11. Triazoles inhibit cholesterol export from lysosomes by binding to NPC1.

    PubMed

    Trinh, Michael N; Lu, Feiran; Li, Xiaochun; Das, Akash; Liang, Qiren; De Brabander, Jef K; Brown, Michael S; Goldstein, Joseph L

    2017-01-03

    Niemann-Pick C1 (NPC1), a membrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly inhibited by itraconazole, a triazole that is used as an antifungal drug [Xu et al. (2010) Proc Natl Acad Sci USA 107:4764-4769]. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.

  12. Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

    PubMed Central

    Grimberg, Brian T; Udomsangpetch, Rachanee; Xainli, Jia; McHenry, Amy; Panichakul, Tasanee; Sattabongkot, Jetsumon; Cui, Liwang; Bockarie, Moses; Chitnis, Chetan; Adams, John; Zimmerman, Peter A; King, Christopher L

    2007-01-01

    Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection. PMID:18092885

  13. Hepatocyte insulin receptor is a calmodulin binding protein and is functionally inhibited by calmidazolium

    SciTech Connect

    Arnold, T.P.; Pollet, R.J.

    1986-05-01

    Insulin-induced autophosphorylation of the insulin receptor and changes in intracellular Ca/sup + +/ have been proposed as possible mediators of insulin action in target tissues. The authors have investigated the association of the 17kD calcium binding protein calmodulin with the insulin receptor solubilized from rat liver plasma membranes. Insulin receptors solubilized in 0.1% Triton X-100 exhibited strong binding to calmodulin-agarose affinity columns in the presence of 100..mu..M calcium and could be eluded with 100..mu..M ethelene glycol-bis (amino ethel ether) Tetra Acetic Acid (EGTA) with an 80% yield in insulin binding activity. In addition, /sup 125/I-Calmodulin was shown to bind to wheat germ agglutinin purified solubilized receptors, was specifically inhibited by EGTA (100 ..mu..M) and/or calmidazolium (10 ..mu..M) and was found to be insulin-dependent (max 10/sup -10/ M insulin). SDS-polyacrylamide gel electrophoresis data suggests that /sup 125/I-calmodulin may be associated with the 92 kD beta-subunit of the insulin receptor, consistent with the cytoplasmic domain of this subunit. While they have confirmed previous reports that the addition of calcium and calmodulin to solubilized insulin receptors preparations produces no demonstrable change in receptor phosphorylation, the addition of the calmodulin inhibitor calmidazolium did show more than 50% inhibition of insulin stimulated receptor phosphorylation, suggesting that a domain of the calmodulin molecule may be very tightly associated with the insulin receptor. These results indicate that calmodulin binds tightly and specifically to the insulin receptor of the hepatocyte and is insulin dependent. The findings also suggest that this interaction may be functionally significant in mediating insulin-induced receptor phosphorylation as well as other insulin actions. Thus, calmodulin may play a major role as an intracellular contributor to insulin action.

  14. Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

    PubMed

    Paiz-Candia, Bertin; Islas, Angel A; Sánchez-Solano, Alfredo; Mancilla-Simbro, Claudia; Scior, Thomas; Millan-PerezPeña, Lourdes; Salinas-Stefanon, Eduardo M

    2017-02-05

    Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na(+) channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na(+) channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Nav1.4 currents (IC50 =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Nav1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na(+) channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.

  15. Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABP(PM).

    PubMed

    Turcotte, L P; Swenberger, J R; Tucker, M Z; Yee, A J; Trump, G; Luiken, J J; Bonen, A

    2000-07-01

    Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP(PM)) may be a component of this system. To test the hypothesis that FABP(PM) is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP(PM). Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 +/- 1.2 pmol/mg protein/15 sec and 45.6 +/- 2.9 nmol/mg protein/15 min and Km values of 12.8 +/- 3.8 and 18.4 +/- 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP(PM). Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP(PM). Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP(PM) is a component of this process in muscle.

  16. Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles.

    PubMed

    Marsden, Anne E; King, Jessica M; Spies, M Ashley; Kim, Oak K; Yahr, Timothy L

    2016-02-01

    The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on an N-hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel of N-hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC50s) for the tested N-hydroxybenzimidazoles ranged from 8 to 45 μM in DNA-binding assays. Each of the N-hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity of N-hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected by N-hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression in Yersinia pestis, Aeromonas hydrophila, and Vibrio parahaemolyticus were also sensitive to N-hydroxybenzimidazoles. Although ExsA and Y. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of the N-hydroxybenzimidazoles were observed. Site-directed mutagenesis based on in silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA to N-hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.

  17. Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.

    PubMed

    Rittershaus, C W; Thomas, L J; Miller, D P; Picard, M D; Geoghegan-Barek, K M; Scesney, S M; Henry, L D; Sen, A C; Bertino, A M; Hannig, G; Adari, H; Mealey, R A; Gosselin, M L; Couto, M; Hayman, E G; Levin, J L; Reinhold, V N; Marsh, H C

    1999-04-16

    Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.

  18. Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of Poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

    SciTech Connect

    McLick, J.; Bauer, P.I.; Hakam, A.; Kun, E.

    1987-04-21

    The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclie and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to (/sup 14/C) dGMP of DNA to form N/sup 2/-((4-carbamoylphenyl)azo)-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH/sub 2/ moiety of the adduct at the (/sup 32/P) nicotinamide-binding site of the enzyme.

  19. Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection.

    PubMed

    Guo, C T; Wong, C H; Kajimoto, T; Miura, T; Ida, Y; Juneja, L R; Kim, M J; Masuda, H; Suzuki, T; Suzuki, Y

    1998-11-01

    We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 microM to 40 microM). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 microM in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.

  20. Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.

    PubMed Central

    Kazazoglou, T; Schackmann, R W; Fosset, M; Shapiro, B M

    1985-01-01

    As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues. Images PMID:3856274

  1. Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ.

    PubMed

    Verhoog, Nicolette; Allie-Reid, Fatima; Vanden Berghe, Wim; Smith, Carine; Haegeman, Guy; Hapgood, Janet; Louw, Ann

    2014-01-01

    Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ's involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ.

  2. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  3. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  4. FMRFamide: low affinity inhibition of opioid binding to rabbit brain membranes

    SciTech Connect

    Zhu, X.Z.; Raffa, R.B.

    1986-03-05

    FMRFamide (Phe-Met-Arg-Phe-NH/sub 2/) was first isolated from the ganglia of molluscs by Price and Greenberg in 1977. The peptide was subsequently shown to have diverse actions on various types of molluscan and mammalian tissues. The presence of immunoreactive FMRFamide-like material (irFMRF) in multiple areas of rat brain, spinal cord, and gastrointestinal tract suggests that irFMRF may have a physiological role in mammals. Tang, Yang and Costa recently demonstrated that FMRFamide attenuates morphine antinociception in rats and postulated, based on this and several other lines of evidence, that irFMRF might be an endogenous opioid antagonist. In the present study, they tested the ability of FMRFamide to inhibit the binding of opioid receptor ligands to rabbit membrane preparations. FMRFamide inhibited the specific binding of both /sup 3/(H)-dihydromorphine and /sup 3/(H)-ethylketocyclazocine (IC/sub 50/ = 14 ..mu..M and 320 ..mu..M, respectively) in a dose-related manner, suggesting that FMRFamide may affect binding to at least two types of opioid receptors (mu and kappa). These data are consistent with the concept that irFMRF might act as an endogenous opioid antagonist. However, the low affinity of FMRFamide leaves open the possibility of another mechanism of opioid antagonism, such as neuromodulation.

  5. Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity.

    PubMed

    Hayashi, Yuko; Kato, Masaki; Seto, Hirokazu; Yamaguchi, Masamitsu

    2006-07-01

    The Drosophila DNA replication-related element binding factor (dDREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of dDREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the dDREF-induced rough eye phenotype, suggesting that Dll negatively regulates dDREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll to interact with the DNA binding domain (16aa to 125aa) of dDREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this dDREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development.

  6. Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets

    PubMed Central

    Moraes, Leonardo A.; Swales, Karen E.; Wray, Jessica A.; Damazo, Amilcar; Gibbins, Jonathan M.; Warner, Timothy D.

    2007-01-01

    Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) γ, PPARβ, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRα and RXRβ. RXR ligands inhibit platelet aggregation and TXA2 release to ADP and the TXA2 receptors, but only weakly to collagen. ADP and TXA2 both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families. PMID:17213293

  7. Zinc binds to and directly inhibits protein phosphatase 2A in vitro.

    PubMed

    Xiong, Yan; Luo, Dan-Ju; Wang, Xiu-Lian; Qiu, Mei; Yang, Yang; Yan, Xiong; Wang, Jian-Zhi; Ye, Qi-Fa; Liu, Rong

    2015-06-01

    Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn(2+) has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn(2+) on PP2A and their direct interaction in vitro. The results showed that Zn(2+) mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 μmol/L) of Zn(2+) inhibited PP2A directly. Further Zn(2+)-IDA-agarose affinity binding assays showed that Zn(2+) bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn(2+) inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.

  8. Heme from Alzheimer's brain inhibits muscarinic receptor binding via thiyl radical generation.

    PubMed

    Venters, H D; Bonilla, L E; Jensen, T; Garner, H P; Bordayo, E Z; Najarian, M M; Ala, T A; Mason, R P; Frey, W H

    1997-08-01

    An endogenous inhibitor (< 3500 Da) of antagonist binding to the muscarinic acetylcholine receptor (mAChR) has been reported to be elevated 3-fold in Alzheimer's disease (AD) brain. This endogenous inhibitor was found to require the presence of reducing agents such as reduced glutathione (GSH) for optimal activity. In the presence of GSH, the inhibitor was shown to generate thiyl radicals which irreversibly inhibited the mAChR. We now report that the inhibitor contains free heme, a well-established source of oxidative stress capable of generating free radicals and causing neurotoxicity. While FeSO4, microperoxidase and hemin all inhibited antagonist binding to the mAChR, only hemin shared the inhibitor's requirement for GSH. Both the free radical scavengers Trolox and Mn2+, and the metal chelator, EDTA, blocked the activity of the endogenous AD inhibitor and of hemin. Heme oxygenase-1 (HO-1) markedly reduced the activity of both the endogenous AD inhibitor and hemin, indicating that the endogenous inhibitor contains heme. Mass spectrometric analysis confirmed the presence of free heme and heme fragments in fractions of the endogenous AD inhibitor. The antioxidants estrogen, vitamin E and vitamin C all protected the mAChR from irreversible inhibition by the endogenous inhibitor or hemin. These antioxidants may function to protect the integrity of the mAChR in vivo and may have therapeutic potential in AD where free heme could be a source of oxidative stress.

  9. Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity.

    PubMed

    Hirose, Fumiko; Ohshima, Nobuko; Kwon, Eun-Jeong; Yoshida, Hideki; Yamaguchi, Masamitsu

    2002-07-01

    Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

  10. Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

    PubMed Central

    2015-01-01

    The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation. PMID:24502234

  11. Epithelial repair is inhibited by an alpha(1,6)-fucose binding lectin.

    PubMed

    Adam, Elizabeth C; Holgate, Stephen T; Lackie, Peter M

    2007-02-01

    The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.

  12. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.

    PubMed

    Zhang, Hai-Nan; Yang, Lina; Ling, Jian-Ya; Czajkowsky, Daniel M; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-Kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-Ce

    2015-12-08

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

  13. Furoates and thenoates inhibit pyruvate dehydrogenase kinase 2 allosterically by binding to its pyruvate regulatory site.

    PubMed

    Masini, Tiziana; Birkaya, Barbara; van Dijk, Simon; Mondal, Milon; Hekelaar, Johan; Jäger, Manuel; Terwisscha van Scheltinga, Anke C; Patel, Mulchand S; Hirsch, Anna K H; Moman, Edelmiro

    2016-01-01

    The last decade has witnessed the reawakening of cancer metabolism as a therapeutic target. In particular, inhibition of pyruvate dehydrogenase kinase (PDK) holds remarkable promise. Dichloroacetic acid (DCA), currently undergoing clinical trials, is a unique PDK inhibitor in which it binds to the allosteric pyruvate site of the enzyme. However, the safety of DCA as a drug is compromised by its neurotoxicity, whereas its usefulness as an investigative tool is limited by the high concentrations required to exert observable effects in cell culture. Herein, we report the identification - by making use of saturation-transfer difference NMR spectroscopy, enzymatic assays and computational methods - of furoate and thenoate derivatives as allosteric pyruvate-site-binding PDK2 inhibitors. This work substantiates the pyruvate regulatory pocket as a druggable target.

  14. Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation.

    PubMed

    Shiba, Emiko; Izawa, Kumi; Kaitani, Ayako; Isobe, Masamichi; Maehara, Akie; Uchida, Koichiro; Maeda, Keiko; Nakano, Nobuhiro; Ogawa, Hideoki; Okumura, Ko; Kitamura, Toshio; Shimizu, Toshiaki; Kitaura, Jiro

    2017-02-17

    LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f(-/-) mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f(-/-) mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo.

  15. Ceramide-CD300f Binding Inhibits Lipopolysaccharide-induced Skin Inflammation*

    PubMed Central

    Shiba, Emiko; Izawa, Kumi; Kaitani, Ayako; Isobe, Masamichi; Maehara, Akie; Uchida, Koichiro; Maeda, Keiko; Nakano, Nobuhiro; Ogawa, Hideoki; Okumura, Ko; Kitamura, Toshio; Shimizu, Toshiaki; Kitaura, Jiro

    2017-01-01

    LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f−/− mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f−/− mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo. PMID:28073916

  16. Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

    PubMed Central

    Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

    1971-01-01

    Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

  17. Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

    PubMed

    Kühn-Wache, Kerstin; Bär, Joachim W; Hoffmann, Torsten; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich

    2011-03-01

    Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P₁-position for proline, alanine, glycine and serine with 5.0 x 10⁵ M⁻¹ s⁻¹, 1.8 x 10⁴ M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P₁-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P₁-position with 4 x 10⁵ M⁻¹ s⁻¹, 9.5 x 10⁵ M⁻¹ s⁻¹ and 2.1 x 10⁵ M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P₄'-P₈'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

  18. Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

    SciTech Connect

    Goldman, M.E.; Pisano, J.J.

    1985-10-07

    Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A/sub 2/ (10 ng/ml) for 15 min caused a 2-fold increase in the K/sub d/ without changing the B/sub max/ compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the K/sub d/ but significantly decreased the B/sub max/ to 71% the value for untreated membranes. (/sup 3/H)Nitrendipine, preincubated with bee venom phospholipase A/sub 2/, was recovered and found to be fully active, indicating that the phospholipase A/sub 2/ did not modify the ligand. It is concluded that phospholipase A/sub 2/ acts on the membrane at or near the (/sup 3/H)nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. 33 references, 3 figures, 1 table.

  19. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    PubMed

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  20. Selective chemical binding enhances cesium tolerance in plants through inhibition of cesium uptake.

    PubMed

    Adams, Eri; Chaban, Vitaly; Khandelia, Himanshu; Shin, Ryoung

    2015-03-05

    High concentrations of cesium (Cs(+)) inhibit plant growth but the detailed mechanisms of Cs(+) uptake, transport and response in plants are not well known. In order to identify small molecules with a capacity to enhance plant tolerance to Cs(+), chemical library screening was performed using Arabidopsis. Of 10,000 chemicals tested, five compounds were confirmed as Cs(+) tolerance enhancers. Further investigation and quantum mechanical modelling revealed that one of these compounds reduced Cs(+) concentrations in plants and that the imidazole moiety of this compound bound specifically to Cs(+). Analysis of the analogous compounds indicated that the structure of the identified compound is important for the effect to be conferred. Taken together, Cs(+) tolerance enhancer isolated here renders plants tolerant to Cs(+) by inhibiting Cs(+) entry into roots via specific binding to the ion thus, for instance, providing a basis for phytostabilisation of radiocesium-contaminated farmland.

  1. Selective chemical binding enhances cesium tolerance in plants through inhibition of cesium uptake

    NASA Astrophysics Data System (ADS)

    Adams, Eri; Chaban, Vitaly; Khandelia, Himanshu; Shin, Ryoung

    2015-03-01

    High concentrations of cesium (Cs+) inhibit plant growth but the detailed mechanisms of Cs+ uptake, transport and response in plants are not well known. In order to identify small molecules with a capacity to enhance plant tolerance to Cs+, chemical library screening was performed using Arabidopsis. Of 10,000 chemicals tested, five compounds were confirmed as Cs+ tolerance enhancers. Further investigation and quantum mechanical modelling revealed that one of these compounds reduced Cs+ concentrations in plants and that the imidazole moiety of this compound bound specifically to Cs+. Analysis of the analogous compounds indicated that the structure of the identified compound is important for the effect to be conferred. Taken together, Cs+ tolerance enhancer isolated here renders plants tolerant to Cs+ by inhibiting Cs+ entry into roots via specific binding to the ion thus, for instance, providing a basis for phytostabilisation of radiocesium-contaminated farmland.

  2. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids (D-aas) are reported to bind to IgE antibodies from people with allergy and asthma. The objectives of this study were to determine if D-aas bind or inhibit IgE binding to peanut allergens, and if they are more effective than L-amino acids (L-aas) in this respect. Several D-aa cocktails...

  3. Antipsychotics inhibit glucose transport: Determination of olanzapine binding site in Staphylococcus epidermidis glucose/H(+) symporter.

    PubMed

    Babkin, Petr; George Thompson, Alayna M; Iancu, Cristina V; Walters, D Eric; Choe, Jun-Yong

    2015-01-01

    The antipsychotic drug olanzapine is widely prescribed to treat schizophrenia and other psychotic disorders. However, it often causes unwanted side effects, including diabetes, due to disruption of insulin-dependant glucose metabolism through a mechanism yet to be elucidated. To determine if olanzapine can affect the first step in glucose metabolism - glucose transport inside cells - we investigated the effect of this drug on the transport activity of a model glucose transporter. The glucose transporter from Staphylococcus epidermidis (GlcPSe) is specific for glucose, inhibited by various human glucose transporter (GLUT) inhibitors, has high sequence and structure homology to GLUTs, and is readily amenable to transport assay, mutagenesis, and computational modeling. We found that olanzapine inhibits glucose transport of GlcPSe with an IC50 0.9 ± 0.1 mM. Computational docking of olanzapine to the GlcPSe structure revealed potential binding sites that were further examined through mutagenesis and transport assay to identify residues important for olanzapine inhibition. These investigations suggest that olanzapine binds in a polar region of the cytosolic part of the transporter, and interacts with residues R129, strictly conserved in all GLUTs, and N136, conserved in only a few GLUTs, including the insulin-responsive GLUT4. We propose that olanzapine inhibits GlcPSe by impeding the alternating opening and closing of the substrate cavity necessary for glucose transport. It accomplishes this by disrupting a key salt bridge formed by conserved residues R129 and E362, that stabilizes the outward-facing conformation of the transporter.

  4. N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

    SciTech Connect

    Ruyechan, W.T. )

    1988-03-01

    The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the {sup 32}P-labeled oligonucleotide dT{sub 10} and native and N-ethylmaleimide-treated ICP8.

  5. Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

    PubMed

    Chinthamani, Sreedevi; Odusanwo, Olutayo; Mondal, Nandini; Nelson, Joel; Neelamegham, Sriram; Baker, Olga J

    2012-04-01

    Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

  6. Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

    SciTech Connect

    Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu

    2011-08-01

    The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPAR{gamma}2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of {beta}-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of {beta}-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

  7. Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses.

    PubMed

    Lin, K c; Ateeq, H S; Hsiung, S H; Chong, L T; Zimmerman, C N; Castro, A; Lee, W C; Hammond, C E; Kalkunte, S; Chen, L L; Pepinsky, R B; Leone, D R; Sprague, A G; Abraham, W M; Gill, A; Lobb, R R; Adams, S P

    1999-03-11

    Integrin alpha4beta1 mediates leukocyte recruitment, activation, mediator release, and apoptosis inhibition, and it plays a central role in inflammatory pathophysiology. High-affinity, selective inhibitors of alpha4beta1, based on the Leu-Asp-Val (LDV) sequence from the alternatively spliced connecting segment-1 (CS-1) peptide of cellular fibronectin, are described that employ a novel N-terminal peptide "cap" strategy. One inhibitor, BIO-1211, was approximately 10(6)-fold more potent than the starting peptide and exhibited tight-binding properties (koff = 1.4 x 10(-4) s-1, KD = 70 pM), a remarkable finding for a noncovalent, small-molecule inhibitor of a protein receptor. BIO-1211 was also 200-fold selective for the activated form of alpha4beta1, and it stimulated expression of ligand-induced epitopes on the integrin beta1 subunit, a property consistent with occupancy of the receptor's ligand-binding site. Pretreatment of allergic sheep with a 3-mg nebulized dose of BIO-1211 inhibited early and late airway responses following antigen challenge and prevented development of nonspecific airway hyperresponsiveness to carbachol. These results show that highly selective and potent small-molecule antagonists can be identified to integrins with primary specificity for peptide domains other than Arg-Gly-Asp (RGD); they confirm the generality of integrins as small molecule targets; and they validate alpha4beta1 as a therapeutic target for asthma.

  8. Metastatin: a hyaluronan-binding complex from cartilage that inhibits tumor growth.

    PubMed

    Liu, N; Lapcevich, R K; Underhill, C B; Han, Z; Gao, F; Swartz, G; Plum, S M; Zhang, L; Green, S J

    2001-02-01

    In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo biological effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability to bind hyaluronan on the target cells. Collectively, these results suggest that Metastatin is an effective antitumor agent that exhibits antiangiogenic activity.

  9. Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition.

    PubMed

    Thakur, Meghna; Seo, Eun Joo; Dever, Thomas E

    2014-02-01

    Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

  10. Phosphorylation of tau at both Thr 231 and Ser 262 is required for maximal inhibition of its binding to microtubules.

    PubMed

    Sengupta, A; Kabat, J; Novak, M; Wu, Q; Grundke-Iqbal, I; Iqbal, K

    1998-09-15

    The paired helical filaments (PHFs) found in Alzheimer's disease (AD) brains are composed primarily of the microtubule-associated protein tau. PHF-tau is in a hyperphosphorylated state and is unable to promote microtubule assembly. We investigated whether the inhibition of tau binding to microtubules is increased when tau is phosphorylated by different kinases in combination with GSK-3. We found that when tau was first phosphorylated by A-kinase, C-kinase, cdk5, or CaM kinase II and then by GSK-3, its binding to microtubules was inhibited by 45, 61, 78, and 79%, respectively. Further, the kinase combinations cdk5/GSK-3 and CaM kinase II/GSK-3 rapidly phosphorylated the sites Thr 231 and Ser 235. When these sites were individually replaced by Ala and the phosphorylation experiments repeated, tau binding to microtubules was inhibited by 54 and 71%, respectively. By comparison, when Ser 262 was replaced by Ala, tau binding to microtubules was inhibited by only 8% after phosphorylation by CaM kinase II. From these observations we estimate that the phosphorylation of Thr 231, Ser 235, and Ser 262 contributes approximately 26, approximately 9, and approximately 33%, respectively, of the overall inhibition of tau binding to microtubules. Together, our results indicate that the binding of tau to microtubules is controlled by the phosphorylation of several sites, among which are Thr 231, Ser 235, and Ser 262.

  11. Inhibition of RNA binding to hepatitis C virus RNA-dependent RNA polymerase: a new mechanism for antiviral intervention

    PubMed Central

    Ahmed-Belkacem, Abdelhakim; Guichou, Jean-François; Brillet, Rozenn; Ahnou, Nazim; Hernandez, Eva; Pallier, Coralie; Pawlotsky, Jean-Michel

    2014-01-01

    The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key target for antiviral intervention. The goal of this study was to identify the binding site and unravel the molecular mechanism by which natural flavonoids efficiently inhibit HCV RdRp. Screening identified the flavonol quercetagetin as the most potent inhibitor of HCV RdRp activity. Quercetagetin was found to inhibit RdRp through inhibition of RNA binding to the viral polymerase, a yet unknown antiviral mechanism. X-ray crystallographic structure analysis of the RdRp-quercetagetin complex identified quercetagetin's binding site at the entrance of the RNA template tunnel, confirming its original mode of action. This antiviral mechanism was associated with a high barrier to resistance in both site-directed mutagenesis and long-term selection experiments. In conclusion, we identified a new mechanism for non-nucleoside inhibition of HCV RdRp through inhibition of RNA binding to the enzyme, a mechanism associated with broad genotypic activity and a high barrier to resistance. Our results open the way to new antiviral approaches for HCV and other viruses that use an RdRp based on RNA binding inhibition, that could prove to be useful in human, animal or plant viral infections. PMID:25053847

  12. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    SciTech Connect

    Kewley, Robyn J. . E-mail: rkewley@csu.edu.au; Whitelaw, Murray L.

    2005-12-09

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer.

  13. Nanoparticles inhibit DNA replication by binding to DNA: modeling and experimental validation.

    PubMed

    Li, Kungang; Zhao, Xiaonan; K Hammer, Brian; Du, Songyan; Chen, Yongsheng

    2013-11-26

    Predictive models are beneficial tools for researchers to use in prioritizing nanoparticles (NPs) for toxicological tests, but experimental evaluation can be time-consuming and expensive, and thus, priority should be given to tests that identify the NPs most likely to be harmful. For characterization of NPs, the physical binding of NPs to DNA molecules is important to measure, as interference with DNA function may be one cause of toxicity. Here, we determined the interaction energy between 12 types of NPs and DNA based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) model and then predicted the affinity of the NPs for DNA. Using the single-molecule imaging technique known as atomic force microscopy (AFM), we experimentally determined the binding affinity of those NPs for DNA. Theoretical predictions and experimental observations of the binding affinity agreed well. Furthermore, the effect of NPs on DNA replication in vitro was investigated with the polymerase chain reaction (PCR) technique. The results showed that NPs with a high affinity for DNA strongly inhibited DNA replication, whereas NPs with low affinity had no or minimal effects on DNA replication. The methodology here is expected to benefit the genotoxicological testing of NPs as well as the design of safe NPs.

  14. Nucleobindin 1 binds to multiple types of pre-fibrillar amyloid and inhibits fibrillization

    PubMed Central

    Bonito-Oliva, Alessandra; Barbash, Shahar; Sakmar, Thomas P.; Graham, W Vallen

    2017-01-01

    During amyloid fibril formation, amyloidogenic polypeptides misfold and self assemble into soluble pre-fibrillar aggregates, i.e., protofibrils, which elongate and mature into insoluble fibrillar aggregates. An emerging class of chaperones, chaperone-like amyloid binding proteins (CLABPs), has been shown to interfere with aggregation of particular misfolded amyloid peptides or proteins. We have discovered that the calcium-binding protein nuclebindin-1 (NUCB1) is a novel CLABP. We show that NUCB1 inhibits aggregation of islet-amyloid polypeptide associated with type 2 diabetes mellitus, a-synuclein associated with Parkinson’s disease, transthyretin V30M mutant associated with familial amyloid polyneuropathy, and Aβ42 associated with Alzheimer’s disease by stabilizing their respective protofibril intermediates. Kinetic studies employing the modeling software AmyloFit show that NUCB1 affects both primary nucleation and secondary nucleation. We hypothesize that NUCB1 binds to the common cross-β-sheet structure of protofibril aggregates to “cap” and stabilize soluble macromolecular complexes. Transmission electron microscopy and atomic force microscopy were employed to characterize the size, shape and volume distribution of multiple sources of NUCB1-capped protofibrils. Interestingly, NUCB1 prevents Aβ42 protofibril toxicity in a cellular assay. NUCB1-stabilized amyloid protofibrils could be used as immunogens to prepare conformation-specific antibodies and as novel tools to develop screens for anti-protofibril diagnostics and therapeutics. PMID:28220836

  15. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  16. PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide.

    PubMed

    Shan, LiXin; Pang, Ling; Zhang, Rumin; Murgolo, Nicholas J; Lan, Hong; Hedrick, Joseph A

    2008-10-10

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) and induces its internalization and degradation. PCSK9 binding to LDLR is mediated through the LDLR epidermal growth factor-like repeat A (EGF-A) domain. We show for the first time that an EGF-A peptide inhibits PCSK9-mediated degradation of LDLR in HepG2 cells. In addition to LDLR, we show that PCSK9 also binds directly to ApoER2 and mouse VLDLR. Importantly, binding of PCSK9 to either LDLR or mouse VLDLR was effectively inhibited by EGF-A while binding to ApoER2 was less affected. In contrast, LDL receptor-associated protein (RAP), which interacts with LDL receptor repeat type A (LA) domains, inhibited PCSK9 binding to ApoER2 with greater efficacy than either LDLR or mVLDLR. These data demonstrate that while PCSK9 binds several receptors via its EGF-A binding domain, additional contacts with other receptor domains are also involved.

  17. Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion

    PubMed Central

    Zhu, Qiuyu; Yamakuchi, Munekazu; Ture, Sara; de la Luz Garcia-Hernandez, Maria; Ko, Kyung Ae; Modjeski, Kristina L.; LoMonaco, Michael B.; Johnson, Andrew D.; O’Donnell, Christopher J.; Takai, Yoshimi; Morrell, Craig N.; Lowenstein, Charles J.

    2014-01-01

    In humans, vWF levels predict the risk of myocardial infarction and thrombosis; however, the factors that influence vWF levels are not completely understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels, though the functional relationship between STXBP5 and vWF is unknown. We hypothesized that STXBP5 inhibits endothelial cell exocytosis. We found that STXBP5 is expressed in human endothelial cells and colocalizes with and interacts with syntaxin 4. In human endothelial cells reduction of STXBP5 increased exocytosis of vWF and P-selectin. Mice lacking Stxbp5 had higher levels of vWF in the plasma, increased P-selectin translocation, and more platelet-endothelial interactions, which suggests that STXBP5 inhibits endothelial exocytosis. However, Stxbp5 KO mice also displayed hemostasis defects, including prolonged tail bleeding times and impaired mesenteric arteriole and carotid artery thrombosis. Furthermore, platelets from Stxbp5 KO mice had defects in platelet secretion and activation; thus, STXBP5 inhibits endothelial exocytosis but promotes platelet secretion. Our study reveals a vascular function for STXBP5, validates the functional relevance of a candidate gene identified by GWAS, and suggests that variation within STXBP5 is a genetic risk for venous thromboembolic disease. PMID:25244095

  18. Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

    PubMed Central

    Hinchliffe, Philip; González, Mariano M.; Mojica, Maria F.; González, Javier M.; Castillo, Valerie; Saiz, Cecilia; Kosmopoulou, Magda; Tooke, Catherine L.; Llarrull, Leticia I.; Mahler, Graciela; Bonomo, Robert A.; Vila, Alejandro J.; Spencer, James

    2016-01-01

    Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Kis of 6–15 µM or 36–84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10–12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26–0.36 µM) than d-BTZs (26–29 µM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120–zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding. PMID:27303030

  19. E1 initiator DNA binding specificity is unmasked by selective inhibition of non-specific DNA binding

    PubMed Central

    Stenlund, Arne

    2003-01-01

    Initiator proteins are critical components of the DNA replication machinery and mark the site of initiation. This activity probably requires highly selective DNA binding; however, many initiators display modest specificity in vitro. We demonstrate that low specificity of the papillomavirus E1 initiator results from the presence of a non-specific DNA-binding activity, involved in melting, which masks the specificity intrinsic to the E1 DNA-binding domain. The viral factor E2 restores specificity through a physical interaction with E1 that suppresses non-specific binding. We propose that this arrangement, where one DNA-binding activity tethers the initiator to ori while another alters DNA structure, is a characteristic of other viral and cellular initiator proteins. This arrangement would provide an explanation for the low selectivity observed for DNA binding by initiator proteins. PMID:12574131

  20. Structural modelling of substrate binding and inhibition in penicillin V acylase from Pectobacterium atrosepticum.

    PubMed

    Avinash, V S; Panigrahi, Priyabrata; Suresh, C G; Pundle, Archana V; Ramasamy, Sureshkumar

    2013-08-09

    Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation.

  1. Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins.

    PubMed Central

    Lü-Lü; Singh, J S; Galperin, M Y; Drake, D; Taylor, K G; Doyle, R J

    1992-01-01

    Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents. Images PMID:1500189

  2. Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite

    PubMed Central

    Clingman, Carina C; Deveau, Laura M; Hay, Samantha A; Genga, Ryan M; Shandilya, Shivender MD; Massi, Francesca; Ryder, Sean P

    2014-01-01

    Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18–22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state. DOI: http://dx.doi.org/10.7554/eLife.02848.001 PMID:24935936

  3. Stereoselective HDAC inhibition from cysteine-derived zinc-binding groups.

    PubMed

    Butler, Kyle V; He, Rong; McLaughlin, Kathryn; Vistoli, Giulio; Langley, Brett; Kozikowski, Alan P

    2009-08-01

    A series of small-molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10, was determined to have IC(50) values below 1 microM. The compounds were also tested in a cellular assay of oxidative stress-induced neurodegeneration. Many of the inhibitors gave near-complete protection against cell death at 10 microM without the neurotoxicity seen with hydroxamic acid-based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc-binding groups (ZBGs). Derivatives of L-cysteine were active in the HDAC inhibition assays, while the derivatives of D-cysteine were inactive. Notably, the finding that both the D- and L-cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.

  4. RNA-binding protein SAMD4 regulates skeleton development through translational inhibition of Mig6 expression

    PubMed Central

    Niu, Ningning; Xiang, Jian-Feng; Yang, Qin; Wang, Lijun; Wei, Zhanying; Chen, Ling-Ling; Yang, Li; Zou, Weiguo

    2017-01-01

    Protein translation regulation has essential roles in inflammatory responses, cancer initiation and the pathogenesis of several neurodegenerative disorders. However, the role of the regulation of protein translation in mammalian skeleton development has been rarely elaborated. Here we report that the lack of the RNA-binding protein sterile alpha motif domain containing protein 4 (SAMD4) resulted in multiple developmental defects in mice, including delayed bone development and decreased osteogenesis. Samd4-deficient mesenchymal progenitors exhibit impaired osteoblast differentiation and function. Mechanism study demonstrates that SAMD4 binds the Mig6 mRNA and inhibits MIG6 protein synthesis. Consistent with this, Samd4-deficient cells have increased MIG6 protein level and knockdown of Mig6 rescues the impaired osteogenesis in Samd4-deficient cells. Furthermore, Samd4-deficient mice also display chondrocyte defects, which is consistent with the regulation of MIG6 protein level by SAMD4. These findings define SAMD4 as a previously unreported key regulator of osteoblastogenesis and bone development, implying that regulation of protein translation is an important mechanism governing skeletogenesis and that control of protein translation could have therapeutic potential in metabolic bone diseases, such as osteoporosis. PMID:28163927

  5. Inhibiting Helicobacter pylori HtrA protease by addressing a computationally predicted allosteric ligand binding site

    PubMed Central

    Perna, Anna Maria; Reisen, Felix; Schmidt, Thomas P.; Geppert, Tim; Pillong, Max; Weisel, Martin; Hoy, Benjamin; Simister, Philip C.; Feller, Stephan M.; Wessler, Silja; Schneider, Gisbert

    2016-01-01

    Helicobacter pylori is associated with inflammatory diseases and can cause gastric cancer and mucosa-associated lymphoma. One of the bacterium’s key proteins is high temperature requirement A (HpHtrA) protein, an extracellular serine protease that cleaves E-cadherin of gastric epithelial cells, which leads to loss of cell-cell adhesion. Inhibition of HpHtrA may constitute an intervention strategy against H. pylori infection. Guided by the computational prediction of hypothetical ligand binding sites on the surface of HpHtrA, we performed residue mutation experiments that confirmed the functional relevance of an allosteric region. We virtually screened for potential ligands addressing this surface cleft located between the catalytic and PDZ1 domains. Our receptor-based computational method represents protein surface pockets in terms of graph frameworks and retrieves small molecules that satisfy the constraints given by the pocket framework. A new chemical entity was identified that blocked E-cadherin cleavage in vitro by direct binding to HpHtrA, and efficiently blocked pathogen transmigration across the gastric epithelial barrier. A preliminary crystal structure of HpHtrA confirms the validity of a comparative “homology” model of the enzyme, which we used for the computational study. The results of this study demonstrate that addressing orphan protein surface cavities of target macromolecules can lead to new bioactive ligands. PMID:26819700

  6. Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

    PubMed Central

    Chen, Yu; Yang, Fan; Zubovic, Lorena; Pavelitz, Tom; Yang, Wen; Godin, Katherine; Walker, Matthew; Zheng, Suxin; Macchi, Paolo; Varani, Gabriele

    2016-01-01

    The RNA Recognition Motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with new specificity would provide valuable tools and an exacting test of our understanding of specificity. We have achieved the first successful re-design of the specificity of an RRM using rational methods and demonstrated re-targeting of activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of miR-21 precursor with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

  7. Binding catalysis and inhibition by metal ions and protons in the enolization of phenylacetylpyrazine.

    PubMed

    Collot, Anne-Gaelle; Courtney, Michael; Coyne, Dara; Eustace, Stephen E; More O'Ferrall, Rory A

    2009-05-01

    A study of the enolization of phenylacetylpyrazine (PzCOCH(2)Ph) catalyzed by acid, base and metal ions in aqueous solution shows, unusually, that metal ions are more effective catalysts than protons, e.g., for zinc k(Zn)/k(H) = 600. Such behavior contrasts with that of the structurally related phenacylpyridine (PyCH(2)COPh) for which k(Zn)/k(H) = 0.0065. To interpret this difference, equilibrium constants for the tautomerization of phenylacetylpyrazine and for binding of protons and metal ions to its keto tautomer and enolate anion have been measured or estimated and are compared with existing measurements for phenacylpyridine. A tautomeric constant, K(E) = 1.2 x 10(-3) (pK(E) = 2.9), is derived by combining forward and reverse rate constants for enolization measured, respectively, by iodination or bromination of the keto tautomer and relaxation of the less stable enol. For the keto tautomer, NMR measurements yield a pK(a) = -0.90 for N-protonation, and spectrophotometric measurements give pK(a) = 11.90 for ionization to an enolate anion. For the enol, pK(a) values of 0.44 and -4.80 for mono- and diprotonation are obtained from the pH profile for ketonization and absorbance measurements for the transient enol reactant. Binding constants for metal ions (Cu(2+), Ni(2+), Zn(2+), Co(2+), and Cd(2+)) are derived from the saturation of their catalysis of the ketonization reaction. It is found that ketonization is efficiently catalyzed by metal ions but inhibited by acid. These findings, and the striking difference from phenacylpyridine, are ascribed to differences in thermodynamic driving force arising from stronger binding of the proton to the more basic pyridine than pyrazine nitrogen atom in both the reactant keto tautomer and in the enaminone or zwitterion product of the rate-determining (proton transfer) step of the enolization.

  8. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    SciTech Connect

    Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine; Gasparutto, Didier; Ravanat, Jean-Luc

    2014-03-28

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

  9. Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

    PubMed

    Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

    2017-03-28

    Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

  10. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    SciTech Connect

    Zhang, Lei; Zhang, Qing; Yang, Yu; Wu, Chuanfang

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  11. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose.

    PubMed

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.

  12. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition

    SciTech Connect

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2016-01-15

    Cardiovirus Leader proteins (L{sub X}) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus L{sub M} structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus L{sub E}, and also larger complexes with L{sub E}:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant L{sub S} and L{sub T} from Theiloviruses. When mutations were introduced to alter the L{sub E} zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on L{sub E} must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by L{sub E}.

  13. RIG-I inhibits pancreatic β cell proliferation through competitive binding of activated Src

    PubMed Central

    Pan, Yi; Li, GuangMing; Zhong, HengGao; Chen, MeiJuan; Chen, TingTing; Gao, LiLi; Wu, HuiWen; Guo, Jun

    2016-01-01

    Nutrition is a necessary condition for cell proliferation, including pancreatic β cells; however, over-nutrition, and the resulting obesity and glucolipotoxicity, is a risk factor for the development of Type 2 diabetes mellitus (DM), and causes inhibition of pancreatic β-cells proliferation and their loss of compensation for insulin resistance. Here, we showed that Retinoic acid (RA)-inducible gene I (RIG-I) responds to nutrient signals and induces loss of β cell mass through G1 cell cycle arrest. Risk factors for type 2 diabetes (e.g., glucolipotoxicity, TNF-α and LPS) activate Src in pancreatic β cells. Elevated RIG-I modulated the interaction of activated Src and STAT3 by competitive binding to STAT3. Elevated RIG-I downregulated the transcription of SKP2, and increased the stability and abundance of P27 protein in a STAT3-dependent manner, which was associated with inhibition of β cell growth elicited by Src. These results supported a role for RIG-I in β cell mass loss under conditions of metabolic surplus and suggested that RIG-I-induced blocking of Src/STAT3 signalling might be involved in G1 phase cycle arrest through the Skp2/P27 pathway in pancreatic β cells. PMID:27349479

  14. Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect*

    PubMed Central

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Boggon, Titus J.; Kang, Sumin; Chen, Jing

    2014-01-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect. PMID:25104357

  15. Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

    PubMed

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J; Shin, Dong M; Khuri, Fadlo R; Boggon, Titus J; Kang, Sumin; Chen, Jing

    2014-09-19

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

  16. Collagen-Binding Peptidoglycans Inhibit MMP Mediated Collagen Degradation and Reduce Dermal Scarring

    PubMed Central

    Snyder, Paul W.; Freeman, Lynetta; Panitch, Alyssa

    2011-01-01

    Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing. PMID:21779387

  17. Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.

    PubMed

    Ciomperlik, Jessica J; Basta, Holly A; Palmenberg, Ann C

    2016-01-01

    Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

  18. Cardiovirus Leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition

    PubMed Central

    Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C.

    2015-01-01

    Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE. PMID:26492198

  19. IgE binding to peanut allergens is inhibited by combined D-aspartic and D-glutamic acids.

    PubMed

    Chung, Si-Yin; Reed, Shawndrika

    2015-01-01

    The objective of this study was to determine if D-amino acids (D-aas) bind and inhibit immunoglobulin E (IgE) binding to peanut allergens. D-aas such as D-Asp (aspartic acid), D-Glu (glutamic acid), combined D-[Asp/Glu] and others were each prepared in a cocktail of 9 other D-aas, along with L-amino acids (L-aas) and controls. Each sample was mixed with a pooled plasma from peanut-allergic donors, and tested by ELISA (enzyme-linked immunosorbent assay) and Western blots for IgE binding to peanut allergens. Results showed that D-[Asp/Glu] (4 mg/ml) inhibited IgE binding (75%) while D-Glu, D-Asp and other D-aas had no inhibitory effect. A higher inhibition was seen with D-[Asp/Glu] than with L-[Asp/Glu]. We concluded that IgE was specific for D-[Asp/Glu], not D-Asp or D-Glu, and that D-[Asp/Glu] was more reactive than was L-[Asp/Glu] in IgE inhibition. The finding indicates that D-[Asp/Glu] may have the potential for removing IgE or reducing IgE binding to peanut allergens in vitro.

  20. Inhibition of AtMYB2 DNA-binding by nitric oxide involves cysteine S-nitrosylation.

    PubMed

    Serpa, Viviane; Vernal, Javier; Lamattina, Lorenzo; Grotewold, Erich; Cassia, Raul; Terenzi, Hernán

    2007-10-05

    Nitric oxide (NO) can influence the transcriptional activity of a wide set of Arabidopsis genes. The aim of the present work was to investigate if NO modifies DNA-binding activity of AtMYB2 (a typical R2R3-MYB from Arabidopsis thaliana), by a posttranslational modification of its conserved Cys53 residue. We cloned a fully active minimal DNA-binding domain of AtMYB2 spanning residues 19-125, hereafter called M2D. In EMSA assays, M2D binds the core binding site 5'-[A]AACC[A]-3'. The NO donors SNP and GSNO inhibit M2D DNA-binding. As expected for a Cys S-nitrosylation, the NO-mediated inhibitory effect was reversed by DTT, and S-nitrosylation of Cys53 in M2D was detected by biotin switch assays. These results demonstrate that the DNA-binding of M2D is inhibited by S-nitrosylation of Cys53 as a consequence of NO action, thus establishing for the first time a relationship between the redox state and DNA-binding in a plant MYB transcription factor.

  1. Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

    PubMed

    Griffiths, Natalie J; Hill, Darryl J; Borodina, Elena; Sessions, Richard B; Devos, Nathalie I; Feron, Christiane M; Poolman, Jan T; Virji, Mumtaz

    2011-12-01

    Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

  2. Dermatophyte-hormone relationships: characterization of progesterone-binding specificity and growth inhibition in the genera Trichophyton and Microsporum.

    PubMed Central

    Clemons, K V; Schär, G; Stover, E P; Feldman, D; Stevens, D A

    1988-01-01

    We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + and - mating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10(-7) to 2 X 10(-7) M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 X 10(-8) M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56 degrees C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding affinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings: DOC and DHT were effective competitors for [3H]progesterone binding. The growth of A. benhamiae + and -, M. canis, and T. rubrum were all inhibited by progesterone in a dose-responsive manner, with 50% inhibition achieved at concentrations of 9.8 x 10(-6), 1.2 x 10(-5), 1.5 x 10(-5), and 2.7 x 10(-6) M. respectively,. PMID:3182998

  3. Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies.

    PubMed

    Navarro-Perán, Enma; Cabezas-Herrera, Juan; Hiner, Alexander N P; Sadunishvili, Tinatin; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

    2005-05-24

    Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.

  4. Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

    PubMed

    Jo, Sooyeon; Bean, Bruce P

    2017-04-01

    Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding.

  5. The Small Molecule R-(-)-β-O-Methylsynephrine Binds to Nucleoporin 153 kDa and Inhibits Angiogenesis

    PubMed Central

    Kim, Nam Hee; Pham, Ngoc Bich; Quinn, Ronald J.; Shim, Joong Sup; Cho, Hee; Cho, Sung Min; Park, Sung Wook; Kim, Jeong Hun; Seok, Seung Hyeok; Oh, Jong-Won; Kwon, Ho Jeong

    2015-01-01

    R-(-)-β-O-methylsynephrine (OMe-Syn) is a naturally occurring small molecule that was identified in a previous screen as an inhibitor of angiogenesis. In this study, we conducted two animal model experiments to investigate the in vivo antiangiogenic activity of OMe-Syn. OMe-Syn significantly inhibited angiogenesis in a transgenic zebrafish model as well as in a mouse retinopathy model. To elucidate the underlying mechanisms responsible for the antiangiogenic activity of OMe-Syn, we used phage display cloning to isolate potential OMe-Syn binding proteins from human cDNA libraries and identified nucleoporin 153 kDa (NUP153) as a primary binding partner of OMe-Syn. OMe-Syn competitively inhibited mRNA binding to the RNA-binding domain of NUP153. Furthermore, depletion of NUP153 in human cells or zebrafish embryos led to an inhibition of angiogenesis, in a manner similar to that seen in response to OMe-Syn treatment. These data suggest that OMe-Syn is a promising candidate for the development of a novel antiangiogenic agent and that inhibition of NUP153 is possibly responsible for the antiangiogenic activity of OMe-Syn. PMID:26221075

  6. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

    PubMed

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E

    2014-04-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  7. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

    PubMed Central

    Shen, Chih-Lung; Gonzalez-Hurtado, Elsie; Zhang, Zhi-Min; Xu, Muyu; Martinez, Ernest; Peng, Chih-Wen; Song, Jikui

    2016-01-01

    Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection. PMID:26845565

  8. Griffithsin binds to the glycosylated proteins (E and prM) of Japanese encephalitis virus and inhibit its infection.

    PubMed

    Ishag, Hassan Z A; Li, Chen; Wang, Fengjuan; Mao, Xiang

    2016-04-02

    Griffithsin (GRFT) is a broad-spectrum antiviral protein against several glycosylated viruses. In our previous publication, we have shown that GRFT exerted antiviral activity against Japanese encephalitis virus (JEV) infection. Herein, we further elucidated the mechanism by which GRFT inhibits JEV infection in BHK-21 cells. In vitro experiments using Pull-down assay and Co-immunoprecipitation (CO-IP) assay showed that GRFT binds to the JEV glycosylated viral proteins, specifically the enveloped (E) and premature (prM) glycoproteins. The binding of GRFT to the JEV was competitively inhibited by increasing concentrations of mannose; in turns abolished anti-JEV activity of GRFT. We suggested that, the binding of GRFT to the glycosylated viral proteins may contribute to its anti-JEV activity. Collectively, our data indicated a possible mechanism by which GRFT exerted its anti-JEV activity. This observation suggests GRFT's potentials in the development of therapeutics against JEV or other flavivirus infection.

  9. RORα Binds to E2F1 To Inhibit Cell Proliferation and Regulate Mammary Gland Branching Morphogenesis

    PubMed Central

    Xiong, Gaofeng

    2014-01-01

    Retinoic acid receptor-related orphan nuclear receptor alpha (RORα) is a potent tumor suppressor that reduces cell proliferation and inhibits tumor growth. However, the molecular mechanism by which it inhibits cell proliferation remains unknown. We demonstrate a noncanonical nuclear receptor pathway in which RORα binds to E2F1 to inhibit cell cycle progression. We showed that RORα bound to the heptad repeat and marked box region of E2F1 and suppressed E2F1-regulated transcription in epithelial cells. Binding of RORα inhibited E2F1 acetylation and its DNA-binding activity by recruiting histone deacetylase 1 (HDAC1) to the protein complexes. Knockdown of HDAC1 or inhibition of HDAC activity at least partially rescued transcription factor activity of E2F1 that was repressed by RORα. Importantly, RORα levels were increased in mammary ducts compared to terminal end buds and inversely correlated with expression of E2F1 target genes and cell proliferation. Silencing RORα in mammary epithelial cells significantly enhanced cell proliferation in the ductal epithelial cells and promoted side branching of the mammary ducts. These results reveal a novel link between RORα and E2F1 in regulating cell cycle progression and mammary tissue morphogenesis. PMID:24891616

  10. Colorectal mucus binds DC-SIGN and inhibits HIV-1 trans-infection of CD4+ T-lymphocytes.

    PubMed

    Stax, Martijn J; Mouser, Emily E I M; van Montfort, Thijs; Sanders, Rogier W; de Vries, Henry J C; Dekker, Henk L; Herrera, Carolina; Speijer, Dave; Pollakis, Georgios; Paxton, William A

    2015-01-01

    Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1-3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

  11. cAMP generation inhibits inositol 1,4,5-trisphosphate binding in rabbit tracheal smooth muscle.

    PubMed

    Schramm, C M; Chuang, S T; Grunstein, M M

    1995-11-01

    Agonist-induced airway contraction involves the generation and subsequent binding of the phosphoinositide-derived second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to its Ca(2+)-mobilizing intracellular receptor. To the extent that regulatory cross-talk is known to exist between different signal transduction pathways, the present study examined whether activation of the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A (PKA) pathway induces altered binding of Ins(1,4,5)P3 to its receptor in membrane homogenates of rabbit tracheal smooth muscle (TSM). In control TSM, monophasic binding curves provided mean +/- SE values for Ins(1,4,5)P3 receptor density (Bmax) and binding affinity (Kd) amounting to 940 +/- 43 fmol/mg protein and 10.7 +/- 1.2 nM, respectively. Relative to control, binding of [3H]Ins(1,4,5)P3 was significantly reduced in paired TSM separately treated with isoproterenol, forskolin, or dibutyryl-cAMP. Ins(1,4,5)P3 binding was inhibited to a level averaging 60% of control binding by maximal concentrations of each agonist, an effect attributed to a reduction in Ins(1,4,5)P3 binding sites rather than altered ligand affinity. Collectively, these findings demonstrate that activation of the cAMP-dependent signaling pathway is associated with inhibition of Ins(1,4,5)P3 receptor binding and implicate a novel mechanism of action of beta-adrenergic agents in preventing and/or reversing airway contraction.

  12. Divalent cations enhance fluoride binding to Streptococcus mutans and Streptococcus sanguinis cells and subsequently inhibit bacterial acid production.

    PubMed

    Domon-Tawaraya, H; Nakajo, K; Washio, J; Ashizawa, T; Ichino, T; Sugawara, H; Fukumoto, S; Takahashi, N

    2013-01-01

    One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

  13. Inhibition of binding of tomato yellow leaf curl virus rep to its replication origin by artificial zinc-finger protein.

    PubMed

    Mori, Tomoaki; Takenaka, Kosuke; Domoto, Fumiya; Aoyama, Yasuhiro; Sera, Takashi

    2013-06-01

    Previously we demonstrated that inhibition of replication-associated protein (Rep) binding to its replication origin by artificial zinc-finger proteins (AZPs) is a powerful method to prevent plant virus infection in vivo. In the present study, we applied the AZP technology to Tomato yellow leaf curl virus (TYLCV), which is a limiting factor in tomato cultivation worldwide. First, we determined 5'-ATCGGTGT ATCGGTGT-3' in the 195-bp intergenic region of the TYLCV-Israel strain, a strain reported first among TYLCV strains, as the Rep-binding site by gel shift assays. We then constructed a 6-finger AZP that bound to a 19-bp DNA including the Rep-binding site. We demonstrated that the binding affinity of the AZP was >1,000-fold greater than that of Rep and that the AZP inhibited Rep binding completely in vitro. Because the binding capability of the AZP was same as that of the AZP previously designed for geminivirus-resistant Arabidopsis thaliana, we predict that the present AZP will prevent TYLCV infection in vivo.

  14. Differentiating a Ligand's Chemical Requirements for Allosteric Interactions from Those for Protein Binding. Phenylalanine Inhibition of Pyruvate Kinase

    SciTech Connect

    Williams,R.; Holyoak, T.; McDonald, G.; Gui, C.; Fenton, A.

    2006-01-01

    The isoform of pyruvate kinase from brain and muscle of mammals (M1-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the L-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M1-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the -carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the -carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 Angstroms structure of M1-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the L-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

  15. Mercury(II) binds to both of chymotrypsin's histidines, causing inhibition followed by irreversible denaturation/aggregation.

    PubMed

    Stratton, Amanda; Ericksen, Matthew; Harris, Travis V; Symmonds, Nick; Silverstein, Todd P

    2017-02-01

    The toxicity of mercury is often attributed to its tight binding to cysteine thiolate anions in vital enzymes. To test our hypothesis that Hg(II) binding to histidine could be a significant factor in mercury's toxic effects, we studied the enzyme chymotrypsin, which lacks free cysteine thiols; we found that chymotrypsin is not only inhibited, but also denatured by Hg(II). We followed the aggregation of denatured enzyme by the increase in visible absorbance due to light scattering. Hg(II)-induced chymotrypsin precipitation increased dramatically above pH 6.5, and free imidazole inhibited this precipitation, implicating histidine-Hg(II) binding in the process of chymotrypsin denaturation/aggregation. Diethylpyrocarbonate (DEPC) blocked chymotrypsin's two histidines (his40 and his57 ) quickly and completely, with an IC50 of 35 ± 6 µM. DEPC at 350 µM reduced the hydrolytic activity of chymotrypsin by 90%, suggesting that low concentrations of DEPC react with his57 at the active site catalytic triad; furthermore, DEPC below 400 µM enhanced the Hg(II)-induced precipitation of chymotrypsin. We conclude that his57 reacts readily with DEPC, causing enzyme inhibition and enhancement of Hg(II)-induced aggregation. Above 500 µM, DEPC inhibited Hg(II)-induced precipitation, and [DEPC] >2.5 mM completely protected chymotrypsin against precipitation. This suggests that his40 reacts less readily with DEPC, and that chymotrypsin denaturation is caused by Hg(II) binding specifically to the his40 residue. Finally, we show that Hg(II)-histidine binding may trigger hemoglobin aggregation as well. Because of results with these two enzymes, we suggest that metal-histidine binding may be key to understanding all heavy metal-induced protein aggregation.

  16. Fyn-phosphorylated PIKE-A binds and inhibits AMPK signaling, blocking its tumor suppressive activity.

    PubMed

    Zhang, S; Qi, Q; Chan, C B; Zhou, W; Chen, J; Luo, H R; Appin, C; Brat, D J; Ye, K

    2016-01-01

    The AMP-activated protein kinase, a key regulator of energy homeostasis, has a critical role in metabolic disorders and cancers. AMPK is mainly regulated by cellular AMP and phosphorylation by upstream kinases. Here, we show that PIKE-A binds to AMPK and blocks its tumor suppressive actions, which are mediated by tyrosine kinase Fyn. PIKE-A directly interacts with AMPK catalytic alpha subunit and impairs T172 phosphorylation, leading to repression of its kinase activity on the downstream targets. Mutation of Fyn phosphorylation sites on PIKE-A, depletion of Fyn, or pharmacological inhibition of Fyn blunts the association between PIKE-A and AMPK, resulting in loss of its inhibitory effect on AMPK. Cell proliferation and oncogenic assays demonstrate that PIKE-A antagonizes tumor suppressive actions of AMPK. In human glioblastoma samples, PIKE-A expression inversely correlates with the p-AMPK levels, supporting that PIKE-A negatively regulates AMPK activity in cancers. Thus, our findings provide additional layer of molecular regulation of the AMPK signaling pathway in cancer progression.

  17. Bovine Lactoferrin Inhibits Toscana Virus Infection by Binding to Heparan Sulphate

    PubMed Central

    Pietrantoni, Agostina; Fortuna, Claudia; Remoli, Maria Elena; Ciufolini, Maria Grazia; Superti, Fabiana

    2015-01-01

    Toscana virus is an emerging sandfly-borne bunyavirus in Mediterranean Europe responsible for neurological diseases in humans. It accounts for about 80% of paediatric meningitis cases during the summer. Despite the important impact of Toscana virus infection-associated disease on human health, currently approved vaccines or effective antiviral treatments are not available. In this research, we have analyzed the effect of bovine lactoferrin, a bi-globular iron-binding glycoprotein with potent antimicrobial and immunomodulatory activities, on Toscana virus infection in vitro. Our results showed that lactoferrin was capable of inhibiting Toscana virus replication in a dose-dependent manner. Results obtained when lactoferrin was added to the cells during different phases of viral infection showed that lactoferrin was able to prevent viral replication when added during the viral adsorption step or during the entire cycle of virus infection, demonstrating that its action takes place in an early phase of viral infection. In particular, our results demonstrated that the anti-Toscana virus action of lactoferrin took place on virus attachment to the cell membrane, mainly through a competition for common glycosaminoglycan receptors. These findings provide further insights on the antiviral activity of bovine lactoferrin. PMID:25643293

  18. 8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode.

    PubMed

    Paoletti, Julie; Assairi, Liliane; Gelin, Muriel; Huteau, Valérie; Nahori, Marie-Anne; Dussurget, Olivier; Labesse, Gilles; Pochet, Sylvie

    2016-11-29

    Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.

  19. Translating slow-binding inhibition kinetics into cellular and in vivo effects

    PubMed Central

    Walkup, Grant K.; You, Zhiping; Ross, Philip L.; Allen, Eleanor K. H.; Daryaee, Fereidoon; Hale, Michael R.; O’Donnell, John; Ehmann, David E.; Schuck, Virna J. A.; Buurman, Ed T.; Choy, Allison L.; Hajec, Laurel; Murphy-Benenato, Kerry; Marone, Valerie; Patey, Sara A.; Grosser, Lena A.; Johnstone, Michele; Walker, Stephen G.; Tonge, Peter J.; Fisher, Stewart L.

    2015-01-01

    Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection. PMID:25894085

  20. Inhibition of Human Metapneumovirus Binding to Heparan Sulfate Blocks Infection in Human Lung Cells and Airway Tissues

    PubMed Central

    Klimyte, Edita M.; Smith, Stacy E.; Oreste, Pasqua; Lembo, David

    2016-01-01

    ABSTRACT Human metapneumovirus (HMPV), a recently discovered paramyxovirus, infects nearly 100% of the world population and causes severe respiratory disease in infants, the elderly, and immunocompromised patients. We previously showed that HMPV binds heparan sulfate proteoglycans (HSPGs) and that HMPV binding requires only the viral fusion (F) protein. To characterize the features of this interaction critical for HMPV binding and the role of this interaction in infection in relevant models, we utilized sulfated polysaccharides, heparan sulfate mimetics, and occluding compounds. Iota-carrageenan demonstrated potent anti-HMPV activity by inhibiting binding to lung cells mediated by the F protein. Furthermore, analysis of a minilibrary of variably sulfated derivatives of Escherichia coli K5 polysaccharide mimicking the HS structure revealed that the highly O-sulfated K5 polysaccharides inhibited HMPV infection, identifying a potential feature of HS critical for HMPV binding. The peptide dendrimer SB105-A10, which binds HS, reduced binding and infection in an F-dependent manner, suggesting that occlusion of HS at the target cell surface is sufficient to prevent infection. HMPV infection was also inhibited by these compounds during apical infection of polarized airway tissues, suggesting that these interactions take place during HMPV infection in a physiologically relevant model. These results reveal key features of the interaction between HMPV and HS, supporting the hypothesis that apical HS in the airway serves as a binding factor during infection, and HS modulating compounds may serve as a platform for potential antiviral development. IMPORTANCE Human metapneumovirus (HMPV) is a paramyxovirus that causes respiratory disease worldwide. It has been previously shown that HMPV requires binding to heparan sulfate on the surfaces of target cells for attachment and infection. In this study, we characterize the key features of this binding interaction using heparan sulfate

  1. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

    PubMed

    Carter, P H; Scherle, P A; Muckelbauer, J K; Voss, M E; Liu, R Q; Thompson, L A; Tebben, A J; Solomon, K A; Lo, Y C; Li, Z; Strzemienski, P; Yang, G; Falahatpisheh, N; Xu, M; Wu, Z; Farrow, N A; Ramnarayan, K; Wang, J; Rideout, D; Yalamoori, V; Domaille, P; Underwood, D J; Trzaskos, J M; Friedman, S M; Newton, R C; Decicco, C P; Muckelbauer, J A

    2001-10-09

    The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

  2. Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins.

    PubMed

    Maddocks, Sarah E; Lopez, Marta Salinas; Rowlands, Richard S; Cooper, Rose A

    2012-03-01

    Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes.

  3. Structure, Sulfatide Binding Properties, and Inhibition of Platelet Aggregation by a Disabled-2 Protein-derived Peptide*

    PubMed Central

    Xiao, Shuyan; Charonko, John J.; Fu, Xiangping; Salmanzadeh, Alireza; Davalos, Rafael V.; Vlachos, Pavlos P.; Finkielstein, Carla V.; Capelluto, Daniel G. S.

    2012-01-01

    Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for αIIbβ3 integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes. PMID:22977233

  4. The venom of Conus pennaceus inhibits the binding of [3H]neuropeptide Y by direct interaction with the radioligand.

    PubMed

    Diallo, B; Vanderheyden, P M; De Backer, J P; Vauquelin, G

    1998-01-01

    The venom from the marine snail Conus pennaceus inhibits the binding of [3H]neuropeptide Y to calf brain membranes (Czerwiec et al., 1996a) and, in the present study, also to rat forebrain membranes. These membranes contain about 80% Y1- and 20% Y2-receptors. The inhibition by the venom was concentration-dependent with an IC50 value of 3.4 micrograms ml-1. However, the venom also inhibited the binding of [3H]neuropeptide Y to the glass fibre filters and to the previously discovered ANPY toxin from the venom of Conus anemone (Czerwiec et al., 1996b). This inhibition was related to the ability of one or more of the venom components to bind directly to the radioligand instead of the initially assumed interaction with the neuropeptide Y receptors present in membrane preparations. The complex with Conus pennaceus venom was not retained by the glass fibre filter during the separation of the bound from the unbound [3H]neuropeptide Y. Gel filtration chromatography and denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the active [3H]neuropeptide Y-binding component is likely a approximately 30 kDa polypeptide. Binding of [3H]neuropeptide Y to the venom component(s) was not displaced by 20 microM of the (1-24) N-terminal and the (25-36) C-terminal neuropeptide Y fragments. It is therefore likely that the recognition of the venom component(s) requires both the C- and the N-terminal segments of the neuropeptide Y molecule.

  5. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    SciTech Connect

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M. . E-mail: laurie.chan@mcgill.ca

    2005-05-15

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl{sub 2}) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl{sub 2} and MeHg on [{sup 3}H]-quinuclidinyl benzilate ([{sup 3}H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B {sub max}) and ligand affinity (K {sub d}). Subsequently, samples were exposed to HgCl{sub 2} or MeHg to derive IC50 values and inhibition constants (K {sub i}). Results demonstrate that HgCl{sub 2} is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [{sup 3}H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.

  6. Substrate-dependent, non-hyperbolic kinetics of pig brain prolyl oligopeptidase and its tight binding inhibition by JTP-4819.

    PubMed

    Venäläinen, Jarkko I; Juvonen, Risto O; Forsberg, Markus M; Garcia-Horsman, Arturo; Poso, Antti; Wallen, Erik A A; Gynther, Jukka; Männistö, Pekka T

    2002-08-01

    Prolyl oligopeptidase (POP) is a cytosolic serine protease that hydrolyses small peptides at the carboxyl end of the proline residue. It has raised pharmaceutical interest, since its inhibitors have been shown to have antiamnesic properties. We studied prolyl oligopeptidase kinetics with two 7-amino-4-methylcoumarin derivatives: Z-Gly-Pro-AMC and Suc-Gly-Pro-AMC. Z-Gly-Pro-AMC was found to obey standard Henri-Michaelis-Menten kinetics with a K(m) of 30+/-3 microM, whereas Suc-Gly-Pro-AMC exhibited substrate inhibition kinetics with K(m) and K(is) of 510+/-150 and 270+/-90 microM, respectively. Autodock simulations revealed that either the succinyl or the AMC-end of Suc-Gly-Pro-AMC may bind to the S'1 subsite of the active site. We believe that non-specifically bound Suc-Gly-Pro-AMC allows the simultaneous binding of second substrate molecule to the active site and this leads in substrate inhibition. In addition, we demonstrated that the inhibition type of a well characterized prolyl oligopeptidase inhibitor, JTP-4819, is competitive tight binding with a K(ic) of 0.045+/-0.008 nM. We suggest that due to the high concentration of prolyl oligopeptidase in the brain (0.12 nmol/g pig brain), the tight binding nature of the inhibition should be considered when using brain homogenate as the enzyme source in prolyl oligopeptidase inhibition measurements. This is of importance in studying structure-activity relationships of potent prolyl oligopeptidase inhibitors.

  7. Resveratrol Inhibits Expression and Binding Activity of the Monocyte Chemotactic Protein-1 Receptor, CCR2, on THP-1 Monocytes

    PubMed Central

    Cullen, John P.; Morrow, David; Jin, Ying; von Offenberg Sweeney, Nicholas; Sitzmann, James V.; Cahill, Paul A.; Redmond, Eileen M.

    2007-01-01

    Monocyte chemotactic protein-1 and its receptor, CCR2, play a key role in atherosclerosis. We determined the effect of the polyphenol, resveratrol, on CCR2 and the mechanisms involved. Resveratrol treatment inhibited 125I-MCP-1 binding to THP-1 cells; 31%, 56%, 84% decrease for 10, 50 and 100 µM resveratrol, in the absence of any effect on receptor affinity. The inhibitory effect of resveratrol on 125I-MCP-1 binding to THP-1 cells and on CCR2 protein expression determined by FACS analysis was attenuated by treatment with L-NAME (NOS inhibitor), PD98059 (MAPK inhibitor) and LY294002 (PI3K inhibitor), whereas neither X/XO (reactive oxygen species generator) nor ICI182780 (estrogen receptor antagonist) had any effect. Concomitant with a decrease in CCR2 protein expression, resveratrol inhibited THP-1 CCR2 mRNA levels, in the absence of any effect on its stability; 26% and 45% inhibition at 10 and 50 µM resveratrol, respectively. This effect was not altered by co-treatment with L-NAME, PD98059 or ICI182780, but was potentiated by LY294002 and X/XO. Conclusions: Resveratrol inhibits monocyte CCR2 binding activity in an NO-, MAPK- and PI3K-dependent manner, whereas it inhibits CCR2 mRNA in an NO- and MAPK-independent, PI3K-dependent manner. These inhibitory effects of resveratrol on chemokine receptor binding and expression may contribute, in part, to its cardiovascular protective activity in vivo. PMID:17499741

  8. Interaction of hirudin with thrombin: Identification of a minimal binding domain of hirudin that inhibits clotting activity

    SciTech Connect

    Mao, S.J.T.; Yates, M.T.; Owen, T.J.; Krstenansky, J.L. )

    1988-10-18

    Hirudin, isolated from the European leech Hirudo medicinalis, is a potent inhibitor of thrombin, forming an almost irreversible thrombin-hirudin complex. Previously, the authors have shown that the carboxyl terminus of hirudin (residues 45-65) inhibits clotting activity and without binding to the catalytic site of thrombin. In the present study, a series of peptides corresponding to this carboxyl-terminal region of hirudin have been synthesized, and their anticoagulant activity and binding properties to thrombin were examined. Binding was assessed by their ability to displace {sup 125}I-hirudin 45-65 from Sepharose-immobilized thrombin and by isolation of peptide-thrombin complexes. They show that the carboxyl-terminal 10 amino acid residues 56-65 (Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln) are minimally required for binding to thrombin and inhibition of clotting. Phe-56 was critical for maintaining anticoagulant activity as demonstrated by the loss of activity when Phe-56 was substituted with D-Phe, Glu, or Leu. In addition, they found that the binding of the carboxyl-terminal peptide of hirudin with thrombin was associated with a significant conformational change of thrombin as judged by circular dichroism. This conformational change might be responsible for the loss of clotting activity of thrombin.

  9. Auto-inhibition of ETV6 (TEL) DNA-binding: appended helices sterically block the ETS domain

    PubMed Central

    Coyne, H. Jerome; De, Soumya; Okon, Mark; Green, Sean M.; Bhachech, Niraja; Graves, Barbara J.; McIntosh, Lawrence P.

    2012-01-01

    ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the auto-inhibition of ETV6 DNA-binding by sequences C-terminal to its ETS domain. The CID (C-terminal inhibitory domain) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and 15N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA-binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity, and complements previous studies of the allosteric mechanism of ETS1 auto-inhibition to reveal both common and divergent features underlying the regulation of DNA-binding by ETS transcription factors. PMID:22584210

  10. The human papillomavirus18 E7 protein inhibits CENP-C binding to α-satellite DNA.

    PubMed

    Yaginuma, Yuji; Yoshimoto, Masafumi; Eguchi, Ayami; Tokuda, Aoi; Takahashi, Shoko

    2015-07-02

    Human papillomavirus (HPV) infection leads to aneuploidy, a numerical chromosomal aberration that is caused by dysregulation of chromosomal segregation. We previously found that the E7 proteins of high-risk HPVs, but not of low-risk HPVs, could bind to centromere protein-C (CENP-C). In this study, we first found that CENP-C could bind centromere α-satellite DNAs using ChIP analysis and HA-tagged CENP-C/nuc transfected 293T cells. We then investigated if HA-CENP-C/nuc binding to α-satellite DNAs was affected by the E7 proteins of high- or low-risk HPVs. We found that transfection of the FLAG tagged HPV18 E7 inhibited the binding of HA-CENP-C/nuc to α-satellite DNAs. This finding was confirmed in HeLa S3 cells transfected with siRNA targeted to HPV18 E7 expression. We therefore speculate that altered function of kinetochores as a result of inhibition of CENP-C and α-satellite DNAs binding may be associated with the chromosomal abnormalities observed in HPV18-positive cancers.

  11. Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes.

    PubMed

    Posner, Mareike G; Upadhyay, Abhishek; Abubaker, Aisha Alsheikh; Fortunato, Tiago M; Vara, Dina; Canobbio, Ilaria; Bagby, Stefan; Pula, Giordano

    2016-02-05

    Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

  12. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650 nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20 μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy.

  13. Natural product (–)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

    PubMed Central

    Lan, Lan; Appelman, Carl; Smith, Amber R.; Yu, Jia; Larsen, Sarah; Marquez, Rebecca T.; Liu, Hao; Wu, Xiaoqing; Gao, Philip; Roy, Anuradha; Anbanandam, Asokan; Gowthaman, Ragul; Karanicolas, John; De Guzman, Roberto N.; Rogers, Steven; Aubé, Jeffrey; Ji, Min; Cohen, Robert S.; Neufeld, Kristi L.; Xu, Liang

    2015-01-01

    Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF-1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (–)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (–)-gossypol with the RNA binding pocket of MSI1. We further showed that (–)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (–)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (–)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy. PMID:25933687

  14. Inhibition of Binding of β2-Glycoprotein 1 to Phosphatidylserine by Polymyxin B, a Lupus-Like Anticoagulant.

    PubMed

    Uchman, Boguslaw; Triplett, Douglas A

    2015-09-01

    Polymyxin B is a cationic peptide that inhibits phospholipid-dependent coagulation tests including activated partial thromboplastin time and to a lesser degree prothrombin time. Thrombin clotting time is insensitive to polymyxin B. β2-glycoprotein 1 (β2GP1) is a cofactor of antiphospholipid antibodies. Antiphospholipid autoantibodies also poses lupus anticoagulant activity through interactions with β2GP1. Using affinity chromatography, polymyxin B can effectively decrease the binding of β2GP1 to immobilize phosphatidylserine. Since then, anticoagulant effect of polymyxin B is most likely due to the binding to negatively charged phospholipids, preventing formation of coagulation complexes.

  15. Inhibition of intramolecular electron transfer in ascorbate oxidase by Ag+: redox state dependent binding.

    PubMed

    Santagostini, Laura; Gullotti, Michele; Hazzard, James T; Maritano, Silvana; Tollin, Gordon; Marchesini, Augusto

    2005-02-01

    Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].

  16. Interferon gamma-induced human guanylate binding protein 1 inhibits mammary tumor growth in mice.

    PubMed

    Lipnik, Karoline; Naschberger, Elisabeth; Gonin-Laurent, Nathalie; Kodajova, Petra; Petznek, Helga; Rungaldier, Stefanie; Astigiano, Simonetta; Ferrini, Silvano; Stürzl, Michael; Hohenadl, Christine

    2010-01-01

    Interferon gamma (IFN-gamma) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-gamma are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-gamma-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-gamma-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-gamma-induced antitumoral defense system.

  17. [Cold inducible RNA-binding protein inhibits hippocampal neuronal apoptosis under hypothermia by regulating redox system].

    PubMed

    Li, Jing-Hui; Zhang, Xue; Meng, Yu; Li, Chang-Sheng; Ji, Hong; Yang, Huan-Min; Li, Shi-Ze

    2015-08-25

    In this study, we intend to confirm our hypothesis that cold inducible RNA-binding protein (CIRP) can inhibit neuronal apoptosis through suppressing the formation of oxygen free radicals under hypothermia. Primary rat hippocampal neurons were isolated and cultured in vitro, and were divided into five groups: (1) normal control group (37 °C), (2) cells infected by empty viral vector group, (3) CIRP over-expressed group, (4) CIRP knock-down group, and (5) hypothermia control group. Cells in groups 2-5 were cultured under 32 °C, 5% CO2. Apoptosis of hippocampal neurons were detected by Annexin V-FITC/PI staining and flow cytometry; Expression of CIRP was determined by Western blot; Redox-related parameters (T-AOC, GSH-Px, SOD, MDA) were detected by ELISA kits. Results showed that CIRP expression levels were significantly increased (P < 0.01) and the apoptotic rates were significantly decreased (P < 0.01) in hypothermia control group and CIRP over-expressed group when compared with normal control group. On the other hand, the apoptotic rate was significantly increased (P < 0.05) in CIRP knock-down group compared with that in hypothermia control group. The levels of redox parameters in hypothermia control group and CIRP over-expressed group were significantly changed in comparison with those in normal control group, CIRP knock-down group and empty viral vector infected group, respectively (P < 0.05 or P < 0.01). These results suggest that up-regulation of CIRP by hypothermia treatment can protect the neuron from apoptosis through suppressing the formation of oxygen free radicals.

  18. Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

    PubMed Central

    Mechaly, Ariel E.; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M.; Pecorari, Frédéric

    2014-01-01

    Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general. PMID:24823716

  19. 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

    2015-10-05

    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.

  20. Caffeic acid phenethyl ester downregulates phospholipase D1 via direct binding and inhibition of NFκB transactivation

    SciTech Connect

    Park, Mi Hee; Kang, Dong Woo; Jung, Yunjin; Choi, Kang-Yell; Min, Do Sik

    2013-12-06

    Highlights: •We found CAFÉ, a natural product that suppresses expression and activity of PLD1. •CAPE decreased PLD1 expression by inhibiting NFκB transactivation. •CAPE rapidly inhibited PLD activity via its binding to a Cys837 of PLD1. •PLD1 downregulation by CAPE inhibited invasion and proliferation of glioma cells. -- Abstract: Upregulation of phospholipase D (PLD) is functionally linked with oncogenic signals and tumorigenesis. Caffeic acid phenethyl ester (CAPE) is an active compound of propolis extract that exhibits anti-proliferative, anti-inflammatory, anti-oxidant, and antineoplastic properties. In this study, we demonstrated that CAPE suppressed the expression of PLD1 at the transcriptional level via inhibition of binding of NFκB to PLD1 promoter. Moreover, CAPE, but not its analogs, bound to a Cys837 residue of PLD1 and inhibited enzymatic activity of PLD. CAPE also decreased activation of matrix metalloproteinases-2 induced by phosphatidic acid, a product of PLD activity. Ultimately, CAPE-induced downregulation of PLD1 suppressed invasion and proliferation of glioma cells. Taken together, the results of this study indicate that CAPE might contribute to anti-neoplastic effect by targeting PLD1.

  1. A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.

    PubMed

    Martens, Erik; Leyssen, An; Van Aelst, Ilse; Fiten, Pierre; Piccard, Helene; Hu, Jialiang; Descamps, Francis J; Van den Steen, Philippe E; Proost, Paul; Van Damme, Jo; Liuzzi, Grazia Maria; Riccio, Paolo; Polverini, Eugenia; Opdenakker, Ghislain

    2007-02-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

  2. Cyanidin-3-o-glucoside directly binds to ERα36 and inhibits EGFR-positive triple-negative breast cancer

    PubMed Central

    Yang, Shiping; Ma, Wenqiang; Liu, Mei; Guo, Shichao; Zhan, Jun; Zhang, Hongquan; Tsang, Suk Ying; Zhang, Ziding; Wang, Zhaoyi; Li, Xiru; Guo, Yang-Dong; Li, Xiangdong

    2016-01-01

    Anthocyanins have been shown to inhibit the growth and metastatic potential of breast cancer (BC) cells. However, the effects of individual anthocyanins on triple-negative breast cancer (TNBC) have not yet been studied. In this study, we found that cyanidin-3-o-glucoside (Cy-3-glu) preferentially promotes the apoptosis of TNBC cells, which co-express the estrogen receptor alpha 36 (ERα36) and the epidermal growth factor receptor (EGFR). We demonstrated that Cy-3-glu directly binds to the ligand-binding domain (LBD) of ERα36, inhibits EGFR/AKT signaling, and promotes EGFR degradation. We also confirmed the therapeutic efficacy of Cy-3-glu on TNBC in the xenograft mouse model. Our data indicates that Cy-3-glu could be a novel preventive/therapeutic agent against the TNBC co-expressed ERα36/EGFR. PMID:27655695

  3. Elucidation of different inhibition mechanism of small chemicals on PtdInsP-binding domains using in silico docking experiments.

    PubMed

    Kim, Yonghwan; Yoon, Youngdae

    2014-05-15

    Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3 and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid-protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases.

  4. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    PubMed Central

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data

  5. Inhibition and Larvicidal Activity of Phenylpropanoids from Piper sarmentosum on Acetylcholinesterase against Mosquito Vectors and Their Binding Mode of Interaction

    PubMed Central

    Hematpoor, Arshia; Liew, Sook Yee; Chong, Wei Lim; Azirun, Mohd Sofian; Lee, Vannajan Sanghiran; Awang, Khalijah

    2016-01-01

    Aedes aegypti, Aedes albopictus and Culex quinquefasciatus are vectors of dengue fever and West Nile virus diseases. This study was conducted to determine the toxicity, mechanism of action and the binding interaction of three active phenylpropanoids from Piper sarmentosum (Piperaceae) toward late 3rd or early 4th larvae of above vectors. A bioassay guided-fractionation on the hexane extract from the roots of Piper sarmentosum led to the isolation and identification of three active phenylpropanoids; asaricin 1, isoasarone 2 and trans-asarone 3. The current study involved evaluation of the toxicity and acetylcholinesterase (AChE) inhibition of these compounds against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae. Asaricin 1 and isoasarone 2 were highly potent against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus larvae causing up to 100% mortality at ≤ 15 μg/mL concentration. The ovicidal activity of asaricin 1, isoasarone 2 and trans-asarone 3 were evaluated through egg hatching. Asaricin 1 and isoasarone 2 showed potent ovicidal activity. Ovicidal activity for both compounds was up to 95% at 25μg/mL. Asaricin 1 and isoasarone 2 showed strong inhibition on acetylcholinesterase with relative IC50 values of 0.73 to 1.87 μg/mL respectively. These findings coupled with the high AChE inhibition may suggest that asaricin 1 and isoasarone 2 are neuron toxic compounds toward Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. Further computational docking with Autodock Vina elaborates the possible interaction of asaricin 1 and isoasarone 2 with three possible binding sites of AChE which includes catalytic triads (CAS: S238, E367, H480), the peripheral sites (PAS: E72, W271) and anionic binding site (W83). The binding affinity of asaricin 1 and isoasarone 2 were relatively strong with asaricin 1 showed a higher binding affinity in the anionic pocket. PMID:27152416

  6. Inhibition of p53-dependent transcription by BOX-I phospho-peptide mimetics that bind to p300

    PubMed Central

    Dornan, David; Hupp, Ted R.

    2001-01-01

    The N-terminal BOX-I domain of p53 containing a docking site for the negative regulator MDM2 and the positive effector p300, harbours two recently identified phosphorylation sites at Thr18 or Ser20 whose affect on p300 is undefined. Biochemical assays demonstrate that although MDM2 binding is inhibited by these phosphorylations, p300 binding is strikingly stabilized by Thr18 or Ser20 phosphorylation. Introducing EGFP-BOX-I domain peptides with an aspartate substitution at Thr18 or Ser20 induced a significant inhibition of endogenous p53-dependent transcription in cycling cells, in irradiated cells, as well as in cells transiently co-transfected with p300 and p53. In contrast an EGFP-wild-type BOX-I domain peptide stimulated p53 activity via inhibition of MDM2 protein binding. These results suggest that phosphorylation of p53 at Thr18 or Ser20 can activate p53 by stabilizing the p300–p53 complex and also identify a class of small molecular weight ligands capable of selective discrimination between MDM2- and p300-dependent activities. PMID:11258706

  7. Anticonvulsants Based on the α-Substituted Amide Group Pharmacophore Bind to and Inhibit Function of Neuronal Nicotinic Acetylcholine Receptors.

    PubMed

    Krivoshein, Arcadius V

    2016-03-16

    Although the antiepileptic properties of α-substituted lactams, acetamides, and cyclic imides have been known for over 60 years, the mechanism by which they act remains unclear. I report here that these compounds bind to the nicotinic acetylcholine receptor (nAChR) and inhibit its function. Using transient kinetic measurements with functionally active, nondesensitized receptors, I have discovered that (i) α-substituted lactams and cyclic imides are noncompetitive inhibitors of heteromeric subtypes (such as α4β2 and α3β4) of neuronal nAChRs and (ii) the binding affinity of these compounds toward the nAChR correlates with their potency in preventing maximal electroshock (MES)-induced convulsions in mice. Based on the hypothesis that α-substituted amide group is the essential pharmacophore of these drugs, I found and tested a simple compound, 2-phenylbutyramide. This compound indeed inhibits nAChR and shows good anticonvulsant activity in mice. Molecular docking simulations suggest that α-substituted lactams, acetamides, and cyclic imides bind to the same sites on the extracellular domain of the receptor. These new findings indicate that inhibition of brain nAChRs may play an important role in the action of these antiepileptic drugs, a role that has not been previously recognized.

  8. COLCEMID INHIBITION OF CELL GROWTH AND THE CHARACTERIZATION OF A COLCEMID-BINDING ACTIVITY IN SACCHAROMYCES CEREVISIAE

    PubMed Central

    Haber, James E.; Peloquin, John G.; Halvorson, Harlyn O.; Borisy, Gary G.

    1972-01-01

    Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 104 liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits. PMID:4561943

  9. Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

    PubMed Central

    Rodríguez, Luis E.; Curtidor, Hernando; Ocampo, Marisol; Garcia, Javier; Puentes, Alvaro; Valbuena, John; Vera, Ricardo; López, Ramses; Patarroyo, Manuel E.

    2005-01-01

    Receptor–ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented α-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%–85%, suggesting that MSP-3 protein’s role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens. PMID:15987906

  10. Ice binding, recrystallization inhibition, and cryoprotective properties of ice-active substances associated with Antarctic sea ice diatoms.

    PubMed

    Raymond, James A; Knight, Charles A

    2003-04-01

    Extracellular macromolecules associated with Antarctic sea ice diatoms were previously shown to have ice-binding activities. The function of these ice-active substances (IASs) has not been identified. Here we show that two of the IASs have a strong ability to inhibit the recrystallization of ice, possibly signifying a cryoprotectant function. To test this possibility, two species of marine diatom (one Antarctic and one temperate) were subjected to a single freeze-thaw cycle (approximately 20h at -4 or -5 degrees C) in the presence or absence of IAS. Viability, based on a double staining technique, was 15-29% higher in the presence of IAS. Etching of single crystal ice hemispheres grown from dilute IAS solutions indicated that the IASs bind to specific faces of ice and are incorporated into the ice lattice. Together, these results suggest that the IASs acts as a cryoprotectant, probably through some ice-binding mechanism.

  11. A novel injection strategy of flurbiprofen axetil by inhibiting protein binding with 6-methoxy-2-naphthylacetic acid.

    PubMed

    Ogata, Kenji; Takamura, Norito; Tokunaga, Jin; Ikeda, Tetsuya; Setoguchi, Nao; Tanda, Kazuhiro; Yamasaki, Tetsuo; Nishio, Toyotaka; Kawai, Keiichi

    2016-04-01

    Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.

  12. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    NASA Astrophysics Data System (ADS)

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  13. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    PubMed Central

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-01-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as ‘transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs. PMID:27499424

  14. Polypyrimidine tract-binding protein induces p19(Ink4d) expression and inhibits the proliferation of H1299 cells.

    PubMed

    Lin, Shankung; Wang, Ming Jen; Tseng, Kuo-Yun

    2013-01-01

    The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that

  15. Imidazoline/guanidinium binding sites and their relation to inhibition of K(ATP) channels in pancreatic B-cells.

    PubMed

    Rustenbeck, I; Herrmann, C; Ratzka, P; Hasselblatt, A

    1997-09-01

    To elucidate the beta-cytotropic effect of imidazoline compounds their inhibitory effect on ATP-dependent K+ channels (K(ATP) channels) in pancreatic B-cells was compared with their binding to membranes from insulin-secreting HIT T15 cells. K(ATP) channels in inside-out patches from B-cells were closed with the following rank order of efficacy at 10 microM: guanabenz > phentolamine = alinidine > clonidine > idazoxan > rilmenidine = amiloride. The last four compounds achieved an incomplete inhibition only. In contrast to sulfonylureas, the inhibitory action of imidazolines was not enhanced by ADP. With intact cells the site which mediates inhibition is less easily accessible for protonated compounds, suggesting a location at the inner face of the plasma membrane. Competition binding experiments were performed by masking alpha-adrenoceptors and using [3H]clonidine as ligand. Homologous displacement of [3H]clonidine revealed two distinct binding sites in HIT cell membranes characterized by dissociation constants of 38 nM and 4,911 nM and maximal binding capacities of 118 fmol/mg protein and 18 pmol/mg protein. Generally, ligands for I2 imidazoline receptors were more potent than ligands for I1 imidazoline receptors to displace [3H]clonidine from the high affinity site, which does not fit into the current classification of imidazoline receptors. Binding to the second site had affinities in the micromolar range, similar to the concentrations necessary to inhibit K(ATP) channels in B-cells. However, alinidine and phentolamine inhibited K(ATP) channels already at concentrations at which they displaced [3H]clonidine only from the high affinity site, but not yet from the low affinity site. Since the proportion of the low and high affinity site varied in dependence of the competitor, the imidazoline binding sites in HIT cells may not be independent, but may rather represent two interacting or interconvertible sites both of which may be involved in K(ATP) channel closure.

  16. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  17. Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis.

    PubMed

    Duckworth, Carrie A; Guimond, Scott E; Sindrewicz, Paulina; Hughes, Ashley J; French, Neil S; Lian, Lu-Yun; Yates, Edwin A; Pritchard, D Mark; Rhodes, Jonathan M; Turnbull, Jeremy E; Yu, Lu-Gang

    2015-09-15

    Concentrations of circulating galectin-3, a metastasis promoter, are greatly increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these modified heparin derivatives and their ultra-low molecular weight sub-fractions had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives (with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections of the derivatives) abolished the circulating galectin-3-mediated increase in lung metastasis of human melanoma and colon cancer cells in nude mice. Structural analysis using nuclear magnetic resonance and synchrotron radiation circular dichroism spectroscopies showed that the modified heparin derivatives bind to the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified, non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding inhibitors with substantial potential as anti-metastasis/cancer drugs.

  18. Activated platelets inhibit hepatocellular carcinoma cell differentiation and promote tumor progression via platelet-tumor cell binding

    PubMed Central

    Xu, Jingchao; Li, Bing; Liu, Yue-Jian; Cheng, Cheng; Zhou, Chunyan; Zhao, Yongfu; Liu, Yang

    2016-01-01

    Lack of differentiation in hepatocellular carcinoma (HCC) is associated with increased circulating platelet size. We measured platelet activation and plasma adenosine diphosphate (ADP) levels in HCC patients based on differentiation status. Local platelet accumulation and platelet-hepatoma cell binding were measured using immunohistochemistry (IHC) or flow cytometry. Using a xenograft assay in NON/SCID mice, we tested the effects of the anti-platelet drug clopidogrel on platelet activation, platelet infiltration, platelet-tumor cell binding and tumor cell differentiation. HCC patients with poor differentiation status displayed elevated platelet activation and higher ADP levels. Platelets accumulated within poorly differentiated tissues and localized at hepatoma cell membranes. Platelet-tumor cell binding was existed in carcinoma tissues, largely mediated by P-selectin on platelets. NOD/SCID mice with xenograft tumors also exhibited increased platelet activation and platelet-tumor cell binding. Clopidogrel therapy triggered hepatoma cell differentiation by attenuating platelet activation and platelet-tumor cell binding. TCF4 knockdown promoted HepG-2 cell differentiation and inhibited tumor formation, and TCF4 could be the potential downstream target for clopidogrel therapy. PMID:27542264

  19. Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides*

    PubMed Central

    Mitra, Sharmistha; Traughber, C. Alicia; Brannon, Mary K.; Gomez, Stephanie; Capelluto, Daniel G. S.

    2013-01-01

    A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. PMID:23880770

  20. Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore

    PubMed Central

    Chokshi, Rikki H.; Larsen, Aaron T.; Bhayana, Brijesh

    2015-01-01

    Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2ʹ′-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118–128 and 228–248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. TASK-3 pore mutations at residues 122 (L122D, E, or K) and 236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore cannot yet be excluded. PMID:26268529

  1. Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO.

    PubMed

    Chiaravalli, Jeanne; Fontan, Elisabeth; Fsihi, Hafida; Coic, Yves-Marie; Baleux, Françoise; Véron, Michel; Agou, Fabrice

    2011-11-01

    Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.

  2. Inhibition of angiogenesis by vitamin D-binding protein: characterization of anti-endothelial activity of DBP-maf.

    PubMed

    Kalkunte, Satyan; Brard, Laurent; Granai, Cornelius O; Swamy, Narasimha

    2005-01-01

    Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis (IC(50) = 7.8 +/- 0.15 microg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.

  3. Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.

    PubMed

    Zhang, Yingnan; Eigenbrot, Charles; Zhou, Lijuan; Shia, Steven; Li, Wei; Quan, Clifford; Tom, Jeffrey; Moran, Paul; Di Lello, Paola; Skelton, Nicholas J; Kong-Beltran, Monica; Peterson, Andrew; Kirchhofer, Daniel

    2014-01-10

    PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 μm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 μm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the β-strand and a discontinuous short α-helix, and it engaged in the same β-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.

  4. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    PubMed

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  5. Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins.

    PubMed

    Mouna, Lina; Hernandez, Eva; Bonte, Dorine; Brost, Rebekka; Amazit, Larbi; Delgui, Laura R; Brune, Wolfram; Geballe, Adam P; Beau, Isabelle; Esclatine, Audrey

    2016-01-01

    Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.

  6. Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins

    PubMed Central

    Mouna, Lina; Hernandez, Eva; Bonte, Dorine; Brost, Rebekka; Amazit, Larbi; Delgui, Laura R.; Brune, Wolfram; Geballe, Adam P.; Beau, Isabelle; Esclatine, Audrey

    2016-01-01

    ABSTRACT Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus. PMID:26654401

  7. Comparison of Chlorpyrifos-Oxon and Paraoxon Acetylcholinesterase Inhibition Dynamics: Potential role of a peripheral binding site

    SciTech Connect

    Kousba, Ahmed A.; Sultatos, L G.; Poet, Torka S.; Timchalk, Chuck

    2004-08-02

    The primary mechanism of action for organophosphorus (OP) insecticides involves the inhibition of acetylcholinesterase (AChE) by oxygenated metabolites (oxons). This inhibition has been attributed to the phosphorylation of the serine hydroxyl group located in the active site of the AChE molecule. The rate of phosphorylation is described by the bimolecular inhibitory rate constant (ki), which has been utilized for quantification of OP inhibitory capacity. It has been previously proposed that a peripheral binding site exists on the AChE molecule, which when occupied, reduces the capacity of additional oxon molecules to phosphorylate the active site. The objective of the current study was to evaluate the interaction of chlorpyrifos oxon (CPO) and paraoxon (PO) with rat brain AChE using a modified Ellman assay in conjunction with a pharmacodynamic model to further assess the dynamics of AChE inhibition and the potential role of a peripheral binding site. The ki for AChE inhibition determined at oxon concentrations of 5 x 10{sup -4} 100 nM were 0.212 and 0.0216 nM-1h-1 for CPO and PO, respectively. The spontaneous reactivation rates of the inhibited AChE for CPO and PO were 0.087 and 0.078 h-1, respectively. In contrast, the ki estimated at a low oxon concentration (1 pM) were {approx} 1,000 and 10,000 -fold higher than those determined at high CPO and PO concentrations, respectively. At these low concentrations, the ki estimates were approximately similar for both CPO and PO (180 and 250 nM-1h-1, respectively). This implies that at low exposure concentrations, both oxons exhibited similar inhibitory potency in contrast to the marked difference exhibited at higher concentrations, which is consistent with the presence of a peripheral binding site on the AChE enzyme. These results support the potential importance of a secondary binding site associated with AChE kinetics, particularly at low environmentally relevant concentrations.

  8. Inhibition of androgen metabolism and binding by a liposterolic extract of "Serenoa repens B" in human foreskin fibroblasts.

    PubMed

    Sultan, C; Terraza, A; Devillier, C; Carilla, E; Briley, M; Loire, C; Descomps, B

    1984-01-01

    We previously suggested [Steroids 33, (1979) 3; Steroids 37, (1981) 6] that cultured genital skin fibroblasts should prove useful for screening of potential antiandrogens in human and living target cells. "Serenoa repens" lipidic extract (S.R.E.) was recently reported (Br. J. Pharmacol., in press) to inhibit androgen action in animals. The present investigation was designed to study the antiandrogenicity of this compound in human cells: we therefore analyzed the effects of S.R.E. on the intracellular conversion of testosterone (T) to 5 alpha-reduced derivatives, and we investigated interaction of S.R.E. with the intracellular androgen-receptor complex. Since the chemical structure of the active component of S.R.E. is still unknown, results are expressed in U/ml (one unit is defined as the amount of S.R.E. required to inhibit 50% of the specific binding (IC50) of [3H]1881 to rat prostate cytosol). S.R.E. at different dilutions (5.7 to 28.6 U/ml) is added to culture media containing [3H]T or [3H]dihydrotestosterone (DHT) and incubated at 37 degrees C with cultured fibroblasts. 28.6 U/ml S.R.E. significantly alters the formation of DHT and strongly inhibits 3 ketosteroid reductase mediated conversion of DHT to 5 alpha-androstane-3 alpha, 17 beta-diol, characterized radiochemically by thin-layer chromatography. S.R.E. is a good competitor for the whole cell androgen receptor: 7.1 U/ml S.R.E. gives 50% inhibition of the binding of 2 X 10(-9) M [3H]DHT to its receptor. Competitive binding assays after cell fractionation indicate that S.R.E. is less potent in nuclear than in cytosol receptors. Sucrose gradient centrifugation of the radioactive cell lysate of fibroblasts demonstrates that 28.6 U/ml S.R.E. abolishes 70% of the 3.6 S receptor-complex radioactive peak. The present studies show that S.R.E. inhibits 5 alpha-reductase, 3-ketosteroid reductase and receptor binding of androgens in cultured human foreskin fibroblasts. As the search for the ideal antiandrogen

  9. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

    PubMed Central

    Lee, Chan-Soo; Choi, Chang-Ki; Schwartz, Martin Alexander

    2010-01-01

    Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology–pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of ∼0.3 µM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of βPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. PMID:20713598

  10. 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

    2014-09-05

    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.

  11. Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

    PubMed

    Pham, Xuan Hoi; Tuteja, Narendra

    2002-06-07

    Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

  12. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    PubMed Central

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. Catalase<-->Abeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro. PMID:10567208

  13. Sulindac-derived RXRα modulators inhibit cancer cell growth by binding to a novel site of RXRα

    PubMed Central

    Chen, Liqun; Wang, Zhi-Gang; Aleshin, Alexander; Chen, Fan; Chen, Jiebo; Jiang, Fuquan; Alitongbieke, Gulimiran; Zeng, Zhiping; Ma, Yue; Huang, Mingfeng; Zhou, Hu; Cadwell, Gregory; Zheng, Jian-Feng; Huang, Pei-Qiang; Liddington, Robert; Zhang, Xiao-kun; Su, Ying

    2014-01-01

    Summary Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anti-cancer effects of certain non-steroidal anti-inflammatory drugs (NSAIDs), including Sulindac. We report the synthesis and characterization of two new Sulindac analogs, K-8008 and K-8012, which exert improved anti-cancer activities over Sulindac in a RXRα- dependent manner. The new analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as new tRXRα modulators and define a new binding mechanism for regulating the nongenomic action of tRXRα. PMID:24704507

  14. Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor.

    PubMed

    Bracci, L; Antoni, G; Cusi, M G; Lozzi, L; Niccolai, N; Petreni, S; Rustici, M; Santucci, A; Soldani, P; Valensin, P E

    1988-09-01

    It has been reported that binding to muscle nicotinic acetylcholine receptor at the post-synaptic membrane is an important event of the rabies virus neurotropism. The binding site can be located within the 190-203 region of the virus glycoprotein sharing a high degree of homology with the "toxic loop" of the curare-mimetic snake neurotoxins. We have synthesized a tetradecapeptide corresponding to this glycoprotein region and used it, following conjugation with an immunogenic carrier to raise MAbs. We found that some MAbs raised against the peptide were able to recognize both the virus glycoprotein and the snake neurotoxin alpha-bungarotoxin; moreover, they can inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor extracted from the electric organs of Torpedo marmorata. On the basis of this cross-reactivity, we suggest that rabies virus glycoprotein and curare-mimetic snake neurotoxins share three-dimensionally similar structures in order to bind to the nicotinic cholinergic receptor. The potential use of the immunogenic properties of the peptide for the rational design of a synthetic vaccine against rabies is proposed.

  15. Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

    PubMed

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

    2010-02-19

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

  16. Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by Stabilizing a Closed State*♦

    PubMed Central

    Fourati, Zaineb; Ruza, Reinis Reinholds; Laverty, Duncan; Drège, Emmanuelle; Delarue-Cochin, Sandrine; Joseph, Delphine; Koehl, Patrice; Smart, Trevor; Delarue, Marc

    2017-01-01

    Barbiturates induce anesthesia by modulating the activity of anionic and cationic pentameric ligand-gated ion channels (pLGICs). Despite more than a century of use in clinical practice, the prototypic binding site for this class of drugs within pLGICs is yet to be described. In this study, we present the first X-ray structures of barbiturates bound to GLIC, a cationic prokaryotic pLGIC with excellent structural homology to other relevant channels sensitive to general anesthetics and, as shown here, to barbiturates, at clinically relevant concentrations. Several derivatives of barbiturates containing anomalous scatterers were synthesized, and these derivatives helped us unambiguously identify a unique barbiturate binding site within the central ion channel pore in a closed conformation. In addition, docking calculations around the observed binding site for all three states of the receptor, including a model of the desensitized state, showed that barbiturates preferentially stabilize the closed state. The identification of this pore binding site sheds light on the mechanism of barbiturate inhibition of cationic pLGICs and allows the rationalization of several structural and functional features previously observed for barbiturates. PMID:27986812

  17. Inhibition of DNA helicase, ATPase and DNA-binding activities of E. coli RecQ helicase by chemotherapeutic agents.

    PubMed

    Zhang, Bo; Zhang, Ai-hua; Chen, Lei; Xi, Xu Guang

    2008-06-01

    RecQ helicases play an essential role in maintaining genetic integrity in all organisms from Escherichia coli to humans. Defects to these enzymes are responsible for three distinct human diseases: Werner syndrome, Bloom syndrome and Rothmund-Thomson syndrome. All three diseases are characterized by a predisposition to cancer due to increased genomic instability. Previous studies on the effects of non-covalent DNA modifications on the catalytic activity of purified Werner and Bloom DNA helicases have shown that both enzymes have similar sensitivity profiles to these DNA-binding agents and are most strongly inhibited by the minor groove binder distamycin A. In this study, we show that the sensitivity profiles of E. coli RecQ to a number of DNA-binding ligands are different to those observed for WRN and Bloom helicases. These observations may give insights into the differences in molecular mechanisms underlying efficient motor function of RecQ helicases.

  18. Arabidopsis Membrane Steroid Binding Protein 1 Is Involved in Inhibition of Cell ElongationW⃞

    PubMed Central

    Yang, Xiao-Hua; Xu, Zhi-Hong; Xue, Hong-Wei

    2005-01-01

    A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220–amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants. PMID:15608331

  19. Inhibition of metabolism and DNA binding of polycyclic aromatic hydrocarbons (PAHs) by plant phenols in epidermis of SENCAR mice

    SciTech Connect

    Das, M.; Bik, D.P.; Bickers, D.R.; Mukhtar, H.

    1986-03-05

    Naturally occurring plant phenols such as tannic acid (TA), quercetin (QT), myricetin (MY) and anthraflavic acid (AA) have been shown to inhibit the mutagenicity of several bay-region diolepoxides of PAHs. Since skin is a target for PAH carcinogenesis, they investigated the effect of these plant phenols on epidermal aryl hydrocarbon hydroxylase (AHH) activity and the binding of PAHs to DNA in SENCAR mice. Each of the plant phenols tested was found to be an in vitro and in vivo inhibitor of epidermal AHH activity with I/sub 50/ values ranging from 4.4 x 10/sup -5/ - 12.4 x 10/sup -5/M in control and 3-methylcholanthrene (MCA) pretreated skin. On an equimolar basis TA was the most potent inhibitor with a Ki of 81 ..mu..M. Incubation of TA, QT, MY and AA with epidermal microsomes resulted in varying degrees of inhibition of enzyme mediated covalent binding of benzo(a)pyrene (BP) to calf thymus DNA. TA (25 ..mu..M) showed maximum inhibition (64%). A single topical application (12 ..mu..mol) of TA, QT, MY and AA resulted in significant decrease in the binding of BP, BP-7,8-diol and 7,12-dimethylbenz(a)anthracene to epidermal DNA. The formation of (+)-7..beta..,8..cap alpha..-dihydroxy-9..cap alpha..,10..cap alpha..-epoxy-7,8,9,10-tetrahydro-BP-deoxyguanine adduct in epidermis was significantly reduced (62-86%) following topical application of the plant phenols. Their results suggest that some of these plant phenols have substantial though variable potential to modify the risk of PAHs induced skin carcinogenicity.

  20. Soluble CD83 Inhibits T Cell Activation by Binding to the TLR4/MD-2 Complex on CD14+ Monocytes

    PubMed Central

    Horvatinovich, Joe M.; Grogan, Elizabeth W.; Norris, Marcus; Steinkasserer, Alexander; Lemos, Henrique; Mellor, Andrew L.; Tcherepanova, Irina Y.; Nicolette, Charles A.

    2017-01-01

    The transmembrane protein CD83, expressed on APCs, B cells, and T cells, can be expressed as a soluble form generated by alternative splice variants and/or by shedding. Soluble CD83 (sCD83) was shown to be involved in negatively regulating the immune response. sCD83 inhibits T cell proliferation in vitro, supports allograft survival in vivo, prevents corneal transplant rejection, and attenuates the progression and severity of autoimmune diseases and experimental colitis. Although sCD83 binds to human PBMCs, the specific molecules that bind sCD83 have not been identified. In this article, we identify myeloid differentiation factor-2 (MD-2), the coreceptor within the TLR4/MD-2 receptor complex, as the high-affinity sCD83 binding partner. TLR4/MD-2 mediates proinflammatory signal delivery following recognition of bacterial LPSs. However, altering TLR4 signaling can attenuate the proinflammatory cascade, leading to LPS tolerance. Our data show that binding of sCD83 to MD-2 alters this signaling cascade by rapidly degrading IL-1R–associated kinase-1, leading to induction of the anti-inflammatory mediators IDO, IL-10, and PGE2 in a COX-2–dependent manner. sCD83 inhibited T cell proliferation, blocked IL-2 secretion, and rendered T cells unresponsive to further downstream differentiation signals mediated by IL-2. Therefore, we propose the tolerogenic mechanism of action of sCD83 to be dependent on initial interaction with APCs, altering early cytokine signal pathways and leading to T cell unresponsiveness. PMID:28193829

  1. Mixed Inhibition of cPEPCK by Genistein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft

    PubMed Central

    Dhanjal, Jaspreet Kaur; Sundar, Durai

    2015-01-01

    Cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) is a critical enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis. cPEPCK converts oxaloacetic acid (OAA) into phosphoenol pyruvate (PEP) in the presence of GTP. cPEPCK is known to be associated with type 2 diabetes. Genistein is an isoflavone compound that shows anti-diabetic and anti-obesitic properties. Experimental studies have shown a decrease in the blood glucose level in the presence of genistein by lowering the functional activity of cPEPCK, an enzyme of gluconeogenesis. Using computational techniques such as molecular modeling, molecular docking, molecular dynamics simulation and binding free energy calculations, we identified cPEPCK as a direct target of genistein. We studied the molecular interactions of genistein with three possible conformations of cPEPCK—unbound cPEPCK (u_cPEPCK), GTP bound cPEPCK (GTP_cPEPCK) and GDP bound cPEPCK (GDP_cPEPCK). Binding of genistein was also compared with an already known cPEPCK inhibitor. We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA). Our results demonstrate that genistein uses the mechanism of mixed inhibition to block the functional activity of cPEPCK and thus can serve as a potential anti-diabetic and anti-obesity drug candidate. We also identified an extended binding site in the catalytic cleft of cPEPCK which is used by 3-MPA to inhibit cPEPCK non-competitively. We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK. PMID:26528723

  2. Inhibition of fibronectin binding and fibronectin-mediated cell adhesion to collagen by a peptide from the second type I repeat of thrombospondin

    PubMed Central

    1993-01-01

    The platelet and extracellular matrix glycoprotein thrombospondin interacts with various types of cells as both a positive and negative modulator of cell adhesion, motility, and proliferation. These effects may be mediated by binding of thrombospondin to cell surface receptors or indirectly by binding to other extracellular matrix components. The role of peptide sequences from the type I repeats of thrombospondin in its interaction with fibronectin were investigated. Fibronectin bound specifically to the peptide Gly-Gly-Trp-Ser-His-Trp from the second type I repeat of thrombospondin but not to the corresponding peptides from the first or third repeats or flanking sequences from the second repeat. The two Trp residues and the His residue were essential for binding, and the two Gly residues enhanced the affinity of binding. Binding of the peptide and intact thrombospondin to fibronectin were inhibited by the gelatin-binding domain of fibronectin. The peptide specifically inhibited binding of fibronectin to gelatin or type I collagen and inhibited fibronectin-mediated adhesion of breast carcinoma and melanoma cells to gelatin or type I collagen substrates but not direct adhesion of the cells to fibronectin, which was inhibited by the peptide Gly-Arg-Gly-Asp-Ser. Thus, the fibronectin- binding thrombospondin peptide Gly-Gly-Trp-Ser-His-Trp is a selective inhibitor of fibronectin-mediated interactions of cells with collagen in the extracellular matrix. PMID:8468356

  3. Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion.

    PubMed

    Díaz, Diana P; Ocampo, Marisol; Varela, Yahson; Curtidor, Hernando; Patarroyo, Manuel A; Patarroyo, Manuel E

    2017-02-15

    This study was aimed at characterising the PPE7 protein from the PE/PPE protein family. The presence and transcription of the rv0354c gene in the Mycobacterium tuberculosis complex was determined and the subcellular localisation of the PPE7 protein on mycobacterial membrane was confirmed by immunoelectron microscope. Two peptides were identified as having high binding activity (HABPs) and were tested in vitro regarding the invasion of Mycobacterium tuberculosis H37Rv. HABP 39224 inhibited invasion in A549 epithelial cells and U937 macrophages by more than 50%, whilst HABP 39225 inhibited invasion by 40% in U937 cells. HABP 39224, located in the protein's C-terminal region, has a completely conserved amino acid sequence in M. tuberculosis complex species and could be selected as a base peptide when designing a subunit-based, anti-tuberculosis vaccine.

  4. Inhibition of /sup 22/Na influx by tricyclic and tetracyclic antidepressants and binding of (/sup 3/H)imipramine in bovine adrenal medullary cells

    SciTech Connect

    Arita, M.; Wada, A.; Takara, H.; Izumi, F.

    1987-10-01

    In bovine adrenal medullary cells we investigated the effects of antidepressants on ionic channels and secretion of catecholamines. Tricyclic (imipramine, amitriptyline and nortriptyline) and tetracyclic (maprotiline and mianserin) antidepressants inhibited carbachol-induced influx of /sup 22/Na, /sup 45/Ca and secretion of catecholamines (IC50, 14-96 microM). Influx of /sup 22/Na, /sup 45/Ca and secretion of catecholamines due to veratridine also were inhibited by these drugs (IC50, 10-17 microM). However, antidepressants did not suppress high concentration of K-induced 45Ca influx and catecholamine secretion, suggesting that antidepressants do not inhibit voltage-dependent Ca channels. (/sup 3/H)Imipramine bound specifically to adrenal medullary cells. Binding was saturable, reversible and with two different equilibrium dissociation constants (13.3 and 165.0 microM). Tricyclic and tetracyclic antidepressants competed for the specific binding of (/sup 3/H)imipramine at the same concentrations as they inhibited /sup 22/Na influx caused by carbachol or veratridine. Carbachol, d-tubocurarine, hexamethonium, tetrodotoxin, veratridine and scorpion venom did not inhibit the specific binding of (/sup 3/H)imipramine. These results suggest that tricyclic and tetracyclic antidepressants bind to two populations of binding sites which are functionally associated with nicotinic receptor-associated ionic channels and with voltage-dependent Na channels, and inhibit Na influx. Inhibition of Na influx leads to the reduction of Ca influx and catecholamine secretion caused by carbachol or veratridine.

  5. Mechanical Strength and Inhibition of the Staphylococcus aureus Collagen-Binding Protein Cna

    PubMed Central

    Herman-Bausier, Philippe; Valotteau, Claire; Pietrocola, Giampiero; Rindi, Simonetta; Alsteens, David; Foster, Timothy J.

    2016-01-01

    ABSTRACT The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN), consistent with the high-affinity “collagen hug” mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation. PMID:27795393

  6. Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity.

    PubMed

    Brkljacić, J; Vojnović Milutinović, D; Dundjerski, J; Matić, G

    2004-05-01

    The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.

  7. Kisspeptin-10 inhibits cell migration in vitro via a receptor-GSK3 beta-FAK feedback loop in HTR8SVneo cells.

    PubMed

    Roseweir, A K; Katz, A A; Millar, R P

    2012-05-01

    Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser9 (which inhibits activity), with a 3-fold increase at 5 min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10min. Phosphorylation of focal adhesion kinase (FAK) at Tyr925 increased 3-fold at 10 min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of G(q/11), Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell-cell adhesion and Tyr925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2-p90rsk-GSK3 beta-FAK feedback interactions.

  8. The binding of human low-density lipoproteins to the surface of schistosomula of Schistosoma mansoni is inhibited by polyanions and reduces the binding of anti-schistosomal antibodies.

    PubMed Central

    Chiang, C. P.; Caulfield, J. P.

    1989-01-01

    Host molecules such as serum lipoproteins, blood group glycolipids, and histocompatibility antigens may bind to schistosomes and thereby prevent immune recognition of the parasite. This study examines the kinetics of lipoprotein binding, the ability of polyanions to inhibit lipoprotein binding, the binding of anti-schistosomal antibodies to worms that have previously bound low-density lipoprotein (LDL), and the distribution of lipoproteins bound to the parasites. Lipoproteins in human serum (HS) and purified LDL, very low-density lipoprotein (VLDL), and apolipoprotein B (apo B) in defined media were demonstrated on the surface of schistosomula of Schistosoma mansoni by fluorescence and immunoelectron microscopy using a polyclonal goat anti-human apolipoprotein B antibody (anti-apo B). By fluorophotometric microscopy, lipoprotein binding began within 15 minutes and was largely completed within 3 hours of exposure. Lipoprotein binding saturated at 10% HS or 20 micrograms protein/300 microliters of purified LDL. Suramin inhibited LDL binding by 59% in a dose-dependent fashion. In the absence of LDL in the medium, 2 mM suramin dissociated 41% of bound LDL from the worm surface within 15 minutes and 10 mg/ml heparin dissociated 36%. The binding of human anti-schistosomal antibodies to schistosomula was inhibited by bound LDL. By fluorescence microscopy, serum or purified lipoproteins were distributed over the entire surface of the parasite with focal areas of high intensity. Ultrastructurally, reaction product was seen on the outer leaflet of the outer tegumental membrane and in aggregates and surrounding vesicular structures varying in diameter from 13 to 83 nm. These studies demonstrate that lipoproteins bind to the surface of schistosomula. The binding of lipoproteins is partially inhibited by polyanions, reduces the binding of human anti-schistosomal antibodies, and may help the parasite escape the immune response. Images Figure 1 Figure 9 Figure 10 PMID:2719071

  9. Soyasaponin Ab ameliorates colitis by inhibiting the binding of lipopolysaccharide (LPS) to Toll-like receptor (TLR)4 on macrophages.

    PubMed

    Lee, In-Ah; Park, Young-Jun; Joh, Eun-Ha; Kim, Dong-Hyun

    2011-12-28

    Many clinical studies have shown that daily intake of soybean [ Glycine max (L.) Merr., Fabacease] or its foods may reduce the risk of osteoporosis, heart attack, hyperlipidemia, coronary heart disease, cardiovascular and chronic renal diseases, and cancers, including prostate, colon, and breast cancers. Of the soy constituents, soyasaponins exhibit anti-aging, antioxidant, apoptotic, and anti-inflammatory effects. However, the anti-inflammatory effect of soyasaponin Ab has not been thoroughly studied. Therefore, we investigated its anti-inflammatory effects in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice and lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Soyasaponin Ab inhibited colon shortening, myeloperoxidase activity, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and activation of the transcription factor nuclear factor-κB (NF-κB). Soyasaponin Ab (1, 2, 5, and 10 μM) inhibited the production of NO (IC(50) = 1.6 ± 0.1 μM) and prostaglandin E(2) (IC(50) = 2.0 ± 0.1 ng/mL), the expression of tumor necrosis factor (TNF)-α (IC(50) = 1.3 ± 0.1 ng/mL), interleukin (IL)-1β (IC(50) = 1.5 ± 0.1 pg/mL), and toll-like receptor (TLR)4, and the phosphorylation of interleukin-1 receptor-associated kinase (IRAK)-1 in LPS-stimulated peritoneal macrophages. Soyasaponin Ab weakly inhibited the phosphorylation of ERK, JNK, and p38. Soyasaponin Ab significantly reduced the binding of Alexa-Fluor-594-conjugated LPS to peritoneal macrophages. Soyasaponin Ab did not affect TLR4 expression or LPS-induced NF-κB activation in TLR4 siRNA-treated peritoneal macrophages (knockdown efficiency of TLR4 > 94%). On the basis of these findings, soyasaponin Ab may ameliorate colitis by inhibiting the binding of LPS to TLR4 on macrophages.

  10. Binding of Hepatitis A Virus to its Cellular Receptor 1 Inhibits T-Regulatory Cell Functions in Humans

    PubMed Central

    Manangeeswaran, Mohanraj; Jacques, Jérôme; Tami, Cecilia; Konduru, Krishnamurthy; Amharref, Nadia; Perrella, Oreste; Casasnovas, Jose M.; Umetsu, Dale T.; DeKruyff, Rosemarie H.; Freeman, Gordon J.; Perrella, Alessandro; Kaplan, Gerardo G.

    2012-01-01

    Background & Aims CD4+ T regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. Methods We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. Results Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-β (TGF–β), which limited leukocyte recruitment and survival, and high levels of interleukin-22, which prevented liver damage. Conclusions Interaction between HAV and its receptor HAVCR1 inhibits Treg cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection—a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anti-cancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection. PMID:22430395

  11. Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

    PubMed Central

    Ettenberg, Seth A.; Charlat, Olga; Daley, Michael P.; Liu, Shanming; Vincent, Karen J.; Stuart, Darrin D.; Schuller, Alwin G.; Yuan, Jing; Ospina, Beatriz; Green, John; Yu, Qunyan; Walsh, Renee; Schmitz, Rita; Heine, Holger; Bilic, Sanela; Ostrom, Lance; Mosher, Rebecca; Hartlepp, K. Felix; Zhu, Zhenping; Fawell, Stephen; Yao, Yung-Mae; Stover, David; Finan, Peter M.; Porter, Jeffery A.; Sellers, William R.; Klagge, Ingo M.; Cong, Feng

    2010-01-01

    Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer. PMID:20713706

  12. Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly.

    PubMed

    Hung, Liang-Yi; Chen, Hua-Ling; Chang, Ching-Wen; Li, Bor-Ran; Tang, Tang K

    2004-06-01

    We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.

  13. Electrochemical detection of anti-tau antibodies binding to tau protein and inhibition of GSK-3β-catalyzed phosphorylation.

    PubMed

    Esteves-Villanueva, Jose O; Martic-Milne, Sanela

    2016-03-01

    Tau protein hyperphosphorylation triggers tau aggregation and its toxicity, leading to neuronal death and cell-to-cell toxicity. Hence, inhibition of protein kinases is a viable tool toward reduction of tau toxicity. By targeting various epitopes of Tau441 protein immobilized on Au surface, the protein kinase inhibition by anti-tau antibodies was measured by surface electrochemistry. The electrochemical impedance spectroscopy was used to measure the charge transfer resistance (Rct) of nonphosphorylated tau-Au film (nTau-Au) and compared with the phosphorylated tau-Au film (pTau-Au). The pTau-Au films were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS), which indicated high phosphorus content. The Rct factor was used as the measure of inhibition efficacies by anti-tau antibodies (D8, A10, P262, and Tau46) in addition to antibody formulation intravenous immunoglobulin (IVIG). The Rct factor for pTau-Au in the absence of antibodies was 0.25 ± 0.08, indicating a dramatic decrease in Rct on phosphorylation. The Rct factors for Tau46 and A10 were 0.57 ± 0.22 and 0.65 ± 0.26, respectively, indicating phosphorylation inhibition. All antibodies exhibited similar binding to nTau-Au. The proposed electrochemical assay may be used for detection of other posttranslational modifications.

  14. Cyclic lipopeptide antibiotics bind to the N-terminal domain of the prokaryotic Hsp90 to inhibit the chaperone activity.

    PubMed

    Minagawa, Shun; Kondoh, Yasumitsu; Sueoka, Keigo; Osada, Hiroyuki; Nakamoto, Hitoshi

    2011-04-01

    Chemical arrays were employed to screen ligands for HtpG, the prokaryotic homologue of Hsp (heat-shock protein) 90. We found that colistins and the closely related polymyxin B interact physically with HtpG. They bind to the N-terminal domain of HtpG specifically without affecting its ATPase activity. The interaction caused inhibition of chaperone function of HtpG that suppresses thermal aggregation of substrate proteins. Further studies were performed with one of these cyclic lipopeptide antibiotics, colistin sulfate salt. It inhibited the chaperone function of the N-terminal domain of HtpG. However, it inhibited neither the chaperone function of the middle domain of HtpG nor that of other molecular chaperones such as DnaK, the prokaryotic homologue of Hsp70, and small Hsp. The addition of colistin sulfate salt increased surface hydrophobicity of the N-terminal domain of HtpG and induced oligomerization of HtpG and its N-terminal domain. These structural changes are discussed in relation to the inhibition of the chaperone function.

  15. Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

    PubMed

    Tiago, Teresa; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2004-05-11

    Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

  16. A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

    PubMed Central

    Kuo, Ping-Hsueh; Chang, Pei-Lin; Wang, Wen-Ching; Chuang, Yung-Jen; Chang, Margaret Dah-Tsyr

    2015-01-01

    As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery. PMID:26064887

  17. Synthesis, DNA binding and topoisomerase I inhibition activity of thiazacridine and imidazacridine derivatives.

    PubMed

    Lafayette, Elizabeth Almeida; Vitalino de Almeida, Sinara Mônica; Pitta, Marina Galdino da Rocha; Carneiro Beltrão, Eduardo Isidoro; da Silva, Teresinha Gonçalves; Olímpio de Moura, Ricardo; Pitta, Ivan da Rocha; de Carvalho, Luiz Bezerra; de Lima, Maria do Carmo Alves

    2013-12-06

    Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 10(4) to 6.01 × 10(4) M(-1). UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  18. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner

    SciTech Connect

    Miller, S.D.; Farmer, G.; Prives, C.

    1995-12-01

    This report discusses new findings that the tumor supressor gene product p53 may play a role as a DNA-binding-dependent regulator of DNA replication. The results were obtained using polyomavirus in replication assays. Details regarding effects on cell growth arrest and transcriptional activation are discussed. 61 refs., 7 figs.

  19. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    SciTech Connect

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D.

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  20. Synthesis, antioxidant, enzyme inhibition and DNA binding studies of novel N-benzylated derivatives of sulfonamide

    NASA Astrophysics Data System (ADS)

    Abbas, Aadil; Murtaza, Shahzad; Tahir, Muhammad Nawaz; Shamim, Saima; Sirajuddin, Muhammad; Rana, Usman Ali; Naseem, Khadija; Rafique, Hummera

    2016-08-01

    A series of novel N-benzylated derivatives of sulfonamide were synthesized and characterized by FT-IR, NMR and XRD analysis. The synthesized compounds were assayed for their biological potential. The biological studies involved antioxidant, enzyme inhibition, and DNA interaction studies. Antioxidant potential was investigated by Ferric Reducing Antioxidant Power assay (FRAP) and DPPH free radical scavenging method, the capacity of synthesized compounds to inhibit the enzyme's activity was assayed by using the well-known Elman method whereas DNA interaction studies were carried out with the help UV-Vis absorption titration method. Moreover, a direct correlation between enzyme inhibition activity and concentration of the compounds was observed both in experimental and molecular docking studies. DNA interaction studies of the synthesized compounds showed weak interaction.

  1. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    PubMed

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2012-10-01

    Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains.

  2. The phosphate clamp: sequence selective nucleic acid binding profiles and conformational induction of endonuclease inhibition by cationic Triplatin complexes

    PubMed Central

    Prisecaru, Andreea; Molphy, Zara; Kipping, Ralph G.; Peterson, Erica J.; Qu, Yun; Kellett, Andrew; Farrell, Nicholas P.

    2014-01-01

    The substitution-inert polynuclear platinum(II) complex (PPC) series, [{trans-Pt(NH3)2(NH2(CH2)nNH3)}2-μ-(trans-Pt(NH3)2(NH2(CH2)nNH2)2}](NO3)8, where n = 5 (AH78P), 6 (AH78 TriplatinNC) and 7 (AH78H), are potent non-covalent DNA binding agents where nucleic acid recognition is achieved through use of the ‘phosphate clamp' where the square-planar tetra-am(m)ine Pt(II) coordination units all form bidentate N–O–N complexes through hydrogen bonding with phosphate oxygens. The modular nature of PPC–DNA interactions results in high affinity for calf thymus DNA (Kapp ∼5 × 107 M−1). The phosphate clamp–DNA interactions result in condensation of superhelical and B-DNA, displacement of intercalated ethidium bromide and facilitate cooperative binding of Hoechst 33258 at the minor groove. The effect of linker chain length on DNA conformational changes was examined and the pentane-bridged complex, AH78P, was optimal for condensing DNA with results in the nanomolar region. Analysis of binding affinity and conformational changes for sequence-specific oligonucleotides by ITC, dialysis, ICP-MS, CD and 2D-1H NMR experiments indicate that two limiting modes of phosphate clamp binding can be distinguished through their conformational changes and strongly suggest that DNA condensation is driven by minor-groove spanning. Triplatin-DNA binding prevents endonuclease activity by type II restriction enzymes BamHI, EcoRI and SalI, and inhibition was confirmed through the development of an on-chip microfluidic protocol. PMID:25414347

  3. CsrA Inhibits Translation Initiation of Escherichia coli hfq by Binding to a Single Site Overlapping the Shine-Dalgarno Sequence▿

    PubMed Central

    Baker, Carol S.; Eöry, Lél A.; Yakhnin, Helen; Mercante, Jeffrey; Romeo, Tony; Babitzke, Paul

    2007-01-01

    Csr (carbon storage regulation) of Escherichia coli is a global regulatory system that consists of CsrA, a homodimeric RNA binding protein, two noncoding small RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering this protein, and CsrD, a specificity factor that targets CsrB and CsrC for degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and pgaA by binding to their leader transcripts and preventing ribosome binding. Translation inhibition is thought to contribute to the observed mRNA destabilization. Each of the previously known target transcripts contains multiple CsrA binding sites. A position-specific weight matrix search program was developed using known CsrA binding sites in mRNA. This search tool identified a potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This putative CsrA binding site matched the SELEX-derived binding site consensus sequence in 8 out of 12 positions. Results from gel mobility shift and footprint assays demonstrated that CsrA binds specifically to this site in the hfq leader transcript. Toeprint and cell-free translation results indicated that bound CsrA inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of csrA caused elevated expression of an hfq′-′lacZ translational fusion, while overexpression of csrA inhibited expression of this fusion. We also found that hfq mRNA is stabilized upon entry into stationary-phase growth by a CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first example of a CsrA-regulated gene that contains only one CsrA binding site. PMID:17526692

  4. Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II.

    PubMed

    Schmitt, Paulina; Wilmes, Miriam; Pugnière, Martine; Aumelas, André; Bachère, Evelyne; Sahl, Hans-Georg; Schneider, Tanja; Destoumieux-Garzón, Delphine

    2010-09-17

    Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.

  5. Substrate masking: binding of RNA by EGTA-inactivated micrococcal nuclease results in artifactual inhibition of RNA processing reactions.

    PubMed Central

    Wang, M J; Gegenheimer, P

    1990-01-01

    Inhibition of an RNA processing reaction after treatment with the Ca2(+)-dependent micrococcal nuclease (MN) is often used as a criterion for the presence of a required RNA or ribonucleoprotein component in the system. Following MN digestion, the nuclease is inactivated with EGTA and radiolabeled substrate is added to assay for remaining RNA processing activity. We found previously that inhibition of RNA processing by MN need not involve RNA hydrolysis: EGTA-inactivated MN can suppress RNA processing if the assay is performed in the absence of carrier RNA. We now demonstrate both by native gel electrophoresis and by nitrocellulose filter retention that EGTA-inactivated MN forms a complex with free RNA which can be dissociated by addition of synthetic polynucleotides or heparin. In the absence of Ca2+, nuclease binds to precursor tRNA with an apparent KD congruent to 1.4 x 10(-6) M, comparable to its reported affinity for DNA. In an assay for endonucleolytic tRNA maturation, inactivated MN bound to radiolabeled pre-tRNA physically blocks the sites of endonuclease cleavage and prevents tRNA processing. We call this phenomenon 'substrate masking'. Addition of excess carrier RNA competes with pre-tRNA for MN binding and restores normal processing. Images PMID:2123540

  6. A novel multiplex poliovirus binding inhibition assay applicable for large serosurveillance and vaccine studies, without the use of live poliovirus.

    PubMed

    Schepp, Rutger M; Berbers, Guy A M; Ferreira, José A; Reimerink, Johan H; van der Klis, Fiona R

    2017-03-01

    Large-scale serosurveillance or vaccine studies for poliovirus using the "gold standard" WHO neutralisation test (NT) are very laborious and time consuming. With the polio eradication at hand and with the removal of live attenuated Sabin strains from the oral poliovirus vaccine (OPV), starting with type 2 (as of April 2016), laboratories will need to conform to much more stringent laboratory biosafety regulations when handling live poliovirus strains. In this study, a poliovirus binding inhibition multiplex immunoassay (polio MIA) using inactivated poliovirus vaccine (IPV-Salk) was developed for simultaneous quantification of serum antibodies directed to all three poliovirus types. Our assay shows a good correlation with the NT and an excellent correlation with the ELISA-based binding inhibition assay (POBI). The assay is highly type-specific and reproducible. Additionally, serum sample throughput increases about fivefold relative to NT and POBI and the amount of serum needed is reduced by more than 90%. In conclusion, the polio MIA can be used as a safe and high throughput application, especially for large-scale surveillance and vaccine studies, reducing laboratory time and serum amounts needed.

  7. Meis1 promotes poly (rC)-binding protein 2 expression and inhibits angiotensin II-induced cardiomyocyte hypertrophy.

    PubMed

    Zhang, Yunjiao; Si, Yi; Ma, Nan

    2016-01-01

    The poly(rC)-binding protein 2 (PCBP2) is currently reported to inhibit cardiac hypertrophy. However, how PCBP2 is regulated at transcriptional level remains unknown. Here, we show that Meis1, a PBX1-related homeobox gene, binds to PCBP2 promoter and promotes its transcription. In human failing heart tissues and murine hypertrophic heart tissues, the mRNA and protein levels of Meis1 are markedly downregulated, and the level of Meis1 significantly correlates with levels of Nppa, Myh7, and PCBP2. In neonatal rat cardiomyocytes, angiotensin II (Ang II) treatment induces hypertrophic growth of the cells (increase in cell size, enhanced protein synthesis, and hyperexpression of hypertrophic fetal genes), which are significantly inhibited by Meis1 overexpression or promoted by Meis1 knockdown. Meis1 also reduces Ang II-induced activation of Akt-mTOR pathway. Finally, we show that PCBP2 overexpression rescues the Meis1 effects of Akt-mTOR pathway and hypertrophy of cardiomyocytes. © 2015 IUBMB Life, 68(1):13-22, 2016.

  8. Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.

    PubMed

    Omae, Yosuke; Hanada, Yuichi; Sekimizu, Kazuhisa; Kaito, Chikara

    2013-08-30

    We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

  9. Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

    PubMed Central

    Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

  10. Non-linear slow-binding inhibition of Aerococcus viridans lactate oxidase by Cibacron Blue 3GA.

    PubMed

    Streitenberger, S A; López-Mas, J A; Sánchez-Ferrer, A; García-Carmona, F

    2001-10-01

    Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).

  11. Halide binding and inhibition of laccase copper clusters: the role of reorganization energy.

    PubMed

    Kepp, Kasper P

    2015-01-20

    Laccase-like proteins are multicopper oxidases involved in several biological and industrial processes. Their application is commonly limited due to inhibition by fluoride and chloride, and as-isolated proteins are often substantially activated by heat, suggesting that multiple redox states can complicate characterization. Understanding these processes at the molecular level is thus desirable but theoretically unexplored. This paper reports systematic calculations of geometries, reorganization energies, and ionization energies for all partly oxidized states of the trinuclear copper clusters in realistic models with ∼200 atoms. Corrections for scalar-relativistic effects, dispersion, and thermal effects were estimated. Fluoride, chloride, hydroxide, or water was bound to the T2 copper site of the oxidized resting state, and the peroxo intermediate was also computed for reference. Antiferromagnetic coupling, assigned oxidation states, and general structures were consistent with known spectroscopic data. The computations show that (i) ligands bound to the T2 site substantially increase the reorganization energy of the second reduction of the resting state and reduce the redox potentials, providing a possible mechanism for inhibition; (ii) the reorganization energy is particularly large for F(-) but also high for Cl(-), consistent with the experimental tendency of inhibition; (iii) reduction leads to release of Cl(-) from the T2 site, suggesting a mechanism for heat/reduction activation of laccases by dissociation of inhibiting halides or hydroxide from T2.

  12. A NONSTEADY STATE MODEL FOR THE TIGHT-BINDING INHIBITION OF THYMIDYLATE SYNTHETASE BY 5-FLUOROURACIL

    EPA Science Inventory

    5-Fluorouracil (5_FU) is a widely used chemotherapeutic drug and tratogen that was chosen as a prototypic toxicant to contruct a biologically based dose-resonse (BBDR) model (Setzer et. al., 2001). Part of the BBDR model simulates the inhibition of thymidylate synthetase (TS), a...

  13. Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

    PubMed Central

    Donalisio, Manuela; Rusnati, Marco; Cagno, Valeria; Civra, Andrea; Bugatti, Antonella; Giuliani, Andrea; Pirri, Giovanna; Volante, Marco; Papotti, Mauro; Landolfo, Santo

    2012-01-01

    Respiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC50s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery. PMID:22850525

  14. Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

    PubMed Central

    Johnstone, M. R.; Ross, P. L.; McLaughlin, R. E.; Olivier, N. B.

    2014-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that inhibits a wide range of β-lactamases. These include class A, class C, and some class D enzymes, which erode the activity of β-lactam drugs in multidrug-resistant pathogens like Pseudomonas aeruginosa and Enterobacteriaceae spp. Avibactam is currently in clinical development in combination with the β-lactam antibiotics ceftazidime, ceftaroline fosamil, and aztreonam. Avibactam has the potential to be the first β-lactamase inhibitor that might provide activity against class C-mediated resistance, which represents a growing concern in both hospital- and community-acquired infections. Avibactam has an unusual mechanism of action: it is a covalent inhibitor that acts via ring opening, but in contrast to other currently used β-lactamase inhibitors, this reaction is reversible. Here, we present a high-resolution structure of avibactam bound to a class C β-lactamase, AmpC, from P. aeruginosa that provided insight into the mechanism of both acylation and recyclization in this enzyme class and highlighted the differences observed between class A and class C inhibition. Furthermore, variants resistant to avibactam that identified the residues important for inhibition were isolated. Finally, the structural information was used to predict effective inhibition by sequence analysis and functional studies of class C β-lactamases from a large and diverse set of contemporary clinical isolates (P. aeruginosa and several Enterobacteriaceae spp.) obtained from recent infections to understand any preexisting variability in the binding pocket that might affect inhibition by avibactam. PMID:25022578

  15. FBS or BSA Inhibits EGCG Induced Cell Death through Covalent Binding and the Reduction of Intracellular ROS Production

    PubMed Central

    Zhang, Yin; Xu, Yu-Ying; Sun, Wen-Jie; Zhang, Mo-Han; Zheng, Yi-Fan; Shen, Han-Ming; Yang, Jun

    2016-01-01

    Previously we have shown that (−)-epigallocatechin gallate (EGCG) can induce nonapoptotic cell death in human hepatoma HepG2 cells only under serum-free condition. However, the underlying mechanism for serum in determining the cell fate remains to be answered. The effects of fetal bovine serum (FBS) and its major component bovine serum albumin (BSA) on EGCG-induced cell death were investigated in this study. It was found that BSA, just like FBS, can protect cells from EGCG-induced cell death in a dose-dependent manner. Detailed analysis revealed that both FBS and BSA inhibited generation of ROS to protect against toxicity of EGCG. Furthermore, EGCG was shown to bind to certain cellular proteins including caspase-3, PARP, and α-tubulin, but not LC3 nor β-actin, which formed EGCG-protein complexes that were inseparable by SDS-gel. On the other hand, addition of FBS or BSA to culture medium can block the binding of EGCG to these proteins. In silico docking analysis results suggested that BSA had a stronger affinity to EGCG than the other proteins. Taken together, these data indicated that the protective effect of FBS and BSA against EGCG-induced cell death could be due to (1) the decreased generation of ROS and (2) the competitive binding of BSA to EGCG. PMID:27830147

  16. Herpes virus deneddylases interrupt the cullin-RING ligase neddylation cycle by inhibiting the binding of CAND1.

    PubMed

    Gastaldello, Stefano; Callegari, Simone; Coppotelli, Giuseppe; Hildebrand, Sebastian; Song, Moshi; Masucci, Maria G

    2012-08-01

    The conserved N-terminal domains of the major tegument proteins of herpes viridae encode cysteine proteases with potent ubiquitin and NEDD8-specific deconjugase activity. Here we show that the Epstein-Barr virus-encoded member of this enzyme family, BPLF1, is targeted to cullin-RING ubiquitin ligases (CRLs) via the interaction of the conserved helix-2 with helix-23 of the C-terminal domain (CTD) of cullins, at a site involved in electrostatic interaction with helix-8 of the CRL regulator CAND1. Mutation of the solvent-exposed Asp86 and Asp90 of helix-2 to Arg does not affect the enzymatic activity of BPLF1 but abolishes cullin binding and prevents CRL inactivation. The binding of the catalytically active BPLF1 to cullins inhibits the recruitment of CAND1 to the deneddylated CRLs and promotes the selective degradation of cullins by the proteasome. Cullin proteolysis is rescued by the overexpression of CAND1 or its CTD-binding N-terminal domain. These findings illustrate a new strategy for viral modulation of CRL activity where the combined effects of cullin deneddylation and their targeting for proteasomal degradation drive stable inactivation of the ligases.

  17. Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis.

    PubMed

    Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

    2011-07-01

    Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F(1)F(0)-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F(0), namely oligomycin and N,N'-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F(1), maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F(1) and F(0) domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F(0) proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F(1)F(0)-ATPase oligomycin sensitivity loss.

  18. Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.

    PubMed Central

    Sprott, Kam M; Chumley, Michael J; Hanson, Janean M; Dobrowsky, Rick T

    2002-01-01

    To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis. PMID:12071851

  19. The MC3 receptor binding affinity of melanocortins correlates with the nitric oxide production inhibition in mice brain inflammation model.

    PubMed

    Muceniece, Ruta; Zvejniece, Liga; Liepinsh, Edgars; Kirjanova, Olga; Baumane, Larisa; Petrovska, Ramona; Mutulis, Felikss; Mutule, Ilze; Kalvinsh, Ivars; Wikberg, Jarl E S; Dambrova, Maija

    2006-06-01

    Melanocortins possess strong anti-inflammatory effects acting in the central nervous system via inhibition of the production of nitric oxide (NO) during brain inflammation. To shed more light into the role of melanocortin (MC) receptor subtypes involved we synthesized and evaluated some novel peptides, modified in the melanocyte-stimulating hormone (MSH) core structure, natural MCs and known MC receptor selective peptides - MS05, MS06. Since the study included both selective, high affinity binders and the novel peptides, it was possible to do the correlation analysis of binding activities and the NO induction-related anti-inflammatory effect of the peptides. beta-MSH, gamma1-MSH, gamma2-MSH, alpha-MSH, MS05, Ac-MS06 and Ac-[Ser12]MS06 caused dose dependent inhibition of the lipopolysaccharide (LPS)-induced increase of NO overproduction in the mice forebrain whereas MSH core modified peptides Ac-[Asp9,Ser12]MS06, [Asp9]alpha-MSH and [Asp16]beta-MSH were devoid of this effect in doses up to 10 nmol per mouse. When the minimal effective dose required for inhibition of NO production was correlated with the in vitro binding activity to MC receptor subtypes a strong and significant correlation was found for the MC3 receptor (r = 0.90; p = 0.0008), whereas weak correlation was present for the other receptors. Our results suggest that the MC3 receptor is the major player in mediating the anti-inflammatory activity of MCs in the central nervous system.

  20. Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12).

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

    Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity. Images PMID:6585787

  1. Anti-human HB-EGF monoclonal antibodies inhibiting ectodomain shedding of HB-EGF and diphtheria toxin binding.

    PubMed

    Hamaoka, Miki; Chinen, Ichino; Murata, Takuya; Takashima, Seiji; Iwamoto, Ryo; Mekada, Eisuke

    2010-07-01

    HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.

  2. Slow-Binding Inhibition: A Theoretical and Practical Course for Students

    ERIC Educational Resources Information Center

    Golicnik, Marko; Stojan, Jure

    2004-01-01

    Tyrosinase (EC 1.14.18.1) catalyzes the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) to 2,3,5,6-tetrahydro-5,6-dioxo-1H-indole-2-carboxylate (dopachrome), according to the classical Michaelis-Menten kinetic mechanism. The enzyme is strongly but slowly inhibited by alpha-amino-beta-[N-(3-hydroxy-4-pyridone)] propionic acid (L-mimosine), a…

  3. Expression of CCAAT/Enhancer Binding Protein Beta in Muscle Satellite Cells Inhibits Myogenesis in Cancer Cachexia

    PubMed Central

    Marchildon, François; Lamarche, Émilie; Lala-Tabbert, Neena; St-Louis, Catherine; Wiper-Bergeron, Nadine

    2015-01-01

    Cancer cachexia is a paraneoplastic syndrome that causes profound weight loss and muscle mass atrophy and is estimated to be the cause of up to 30% of cancer deaths. Though the exact cause is unknown, patients with cancer cachexia have increased muscle protein catabolism. In healthy muscle, injury activates skeletal muscle stem cells, called satellite cells, to differentiate and promote regeneration. Here, we provide evidence that this mechanism is inhibited in cancer cachexia due to persistent expression of CCAAT/Enhancer Binding Protein beta (C/EBPβ) in muscle myoblasts. C/EBPβ is a bzip transcription factor that is expressed in muscle satellite cells and is normally downregulated upon differentiation. However, in myoblasts exposed to a cachectic milieu, C/EBPβ expression remains elevated, despite activation to differentiate, resulting in the inhibition of myogenin expression and myogenesis. In vivo, cancer cachexia results in increased number of Pax7+ cells that also express C/EBPβ and the inhibition of normal repair mechanisms. Loss of C/EBPβ expression in primary myoblasts rescues differentiation under cachectic conditions without restoring myotube size, indicating that C/EBPβ is an important inhibitor of myogenesis in cancer cachexia. PMID:26709824

  4. Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.

    PubMed

    Hooker, Erika; Baldwin, Cindy; Roodman, Victoria; Batra, Anupam; Takano, Tomoko; Lemay, Serge

    2017-03-08

    The adapter protein Dok-4 has been reported as both activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4  through an atypical PTB domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both a an interaction-dependent mechanism and a PH domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with  increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.

  5. Merlin inhibits growth hormone-regulated Raf-ERKs pathways by binding to Grb2 protein

    SciTech Connect

    Lim, Jung Yeon; Kim, Hongtae; Jeun, Sin-Soo . E-mail: ssjeun@catholic.ac.kr; Kang, Seok-Gu; Lee, Kyung-Jin

    2006-02-24

    Numerous studies have suggested that the NF2 protein merlin is involved in the regulation of abnormal cell growth and proliferation. In this study, to better understand the merlin's mechanisms that contribute to the inhibition of tumorigenesis, we examined the potential action of merlin on the cell proliferative signaling pathways in response to growth hormone (GH). Merlin effectively attenuated the GH-induced serum response element (SRE) and Elk-1-mediated transcriptional activation, as well as the endogenous SRE-regulated gene c-fos expression in NIH3T3 cells. In addition, merlin prevented the Raf-1 complex activation process, which resulted in the suppression of MAP kinase/ERK, extracellular signal-regulated kinase (ERKs), and Elk-1 phosphorylation, which are the downstream signals of Raf-1. Moreover, it was shown that merlin interacted with endogenous growth factor receptor bound 2 (Grb2) protein and inhibited its expression. These results suggest that merlin contributes, via its protein-to-protein interaction with Grb2 and consequent inhibition of the MAPK pathways, to the regulation of the abnormal cell proliferation, and this provides a further mechanism underlying the tumor suppressor function of merlin.

  6. Cangrelor inhibits the binding of the active metabolites of clopidogrel and prasugrel to P2Y12 receptors in vitro.

    PubMed

    Judge, Heather M; Buckland, Robert J; Jakubowski, Joseph A; Storey, Robert F

    2016-01-01

    Cangrelor is a rapid-acting, direct-binding, and reversible P2Y12 antagonist which has been studied for use during percutaneous coronary intervention (PCI) in patients with or without pretreatment with an oral P2Y12 antagonist. As cangrelor is administered intravenously, it is necessary to switch to an oral P2Y12 antagonist following PCI, such as the thienopyridines clopidogrel, and prasugrel or the non-pyridine ticagrelor. Previous studies have suggested a negative pharmacodynamic interaction between cangrelor and thienopyridines. This in vitro study evaluated the receptor-level interaction between cangrelor and the active metabolite (AM) of clopidogrel or prasugrel by assessing functional P2Y12 receptor number using a (33)P-2MeSADP binding assay. All P2Y12 antagonists studied resulted in strong P2Y12 receptor blockade (cangrelor: 93.6%; clopidogrel AM: 93.0%; prasugrel AM: 97.9%). Adding a thienopyridine AM in the presence of cangrelor strongly reduces P2Y12 receptor blockade by the AM (clopidogrel AM: 7%, prasugrel AM: 3.2%). The thienopyridine AMs had limited ability to compete with cangrelor for binding to P2Y12 (% P2Y12 receptor blockade after co-incubation with cangrelor 1000 nmol/L: 11.7% for clopidogrel AM 3 µmol/L; 34.1% for prasugrel AM 3 µmol/L). In conclusion, in vitro cangrelor strongly inhibits the binding of clopidogrel and prasugrel AMs to the P2Y12 receptor, consistent with the previous observation of a negative pharmacodynamic interaction. Care may need to be taken to not overlap exposure to thienopyridine AMs and cangrelor in order to reduce the risk of thrombotic complications following PCI.

  7. Inhibition of HIV-1 en