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Sample records for matrix proteins catenins

  1. Enhancement of Intervertebral Disc Cell Senescence by WNT/β-Catenin Signaling–Induced Matrix Metalloproteinase Expression

    PubMed Central

    Hiyama, Akihiko; Sakai, Daisuke; Risbud, Makarand V.; Tanaka, Masahiro; Arai, Fumiyuki; Abe, Koichiro; Mochida, Joji

    2013-01-01

    Objective To determine whether intervertebral disc (IVD) cells express β-catenin and to assess the role of the WNT/β-catenin signaling pathway in cellular senescence and aggrecan synthesis. Methods The expression of β-catenin messenger RNA (mRNA) and protein in rat IVD cells was assessed by using several real-time reverse transcription–polymerase chain reaction, Western blot, immunohistochemical, and immunofluorescence analyses. The effect of WNT/β-catenin on nucleus pulposus (NP) cells was examined by transfection experiments, an MTT assay, senescence-associated β-galactosidase staining, a cell cycle analysis, and a transforming growth factor (TGFβ)/bone morphogenetic protein (BMP) pathway–focused microarray analysis. Results We found that β-catenin mRNA and protein were expressed in discs in vivo and that rat NP cells exhibited increased β-catenin mRNA and protein upon stimulation with lithium chloride, a known activator of WNT signaling. LiCl treatment inhibited the proliferation of NP cells in a time- and dose-dependent manner. In addition, there was an increased level of cellular senescence in LiCl-treated cells. Long-term treatment with LiCl induced cell cycle arrest and promoted subsequent apoptosis in NP cells. Activation of WNT/β-catenin signaling also regulated the expression of aggrecan. We also demonstrated that WNT/β-catenin signaling induced the expression of matrix metalloproteinases (MMPs) and TGFβ in NP cells. Conclusion The activation of WNT/β-catenin signaling promotes cellular senescence and may modulate MMP and TGFβ signaling in NP cells. We hypothesize that the activation of WNT/β-catenin signaling may lead to an increased breakdown of the matrix, thereby promoting IVD degeneration. PMID:20533544

  2. Amyloid precursor protein modulates β-catenin degradation

    PubMed Central

    Chen, Yuzhi; Bodles, Angela M

    2007-01-01

    Background The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. Results We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. Conclusion We have provided strong evidence that APP modulates β-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation. PMID:18070361

  3. Connections between cadherin-catenin proteins, spindle misorientation, and cancer

    PubMed Central

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2015-01-01

    Cadherin-catenin mediated adhesion is an important determinant of tissue architecture in multicellular organisms. Cancer progression and maintenance is frequently associated with loss of their expression or functional activity, which not only leads to decreased cell-cell adhesion, but also to enhanced tumor cell proliferation and loss of differentiated characteristics. This review is focused on the emerging implications of cadherin-catenin proteins in the regulation of polarized divisions through their connections with the centrosomes, cytoskeleton, tissue tension and signaling pathways; and illustrates how alterations in cadherin-catenin levels or functional activity may render cells susceptible to transformation through the loss of their proliferation-differentiation balance. PMID:26451345

  4. Regulation of Wnt/β-Catenin Signaling by Protein Kinases

    PubMed Central

    Verheyen, Esther M.; Gottardi, Cara J.

    2011-01-01

    The Wnt/β-catenin signaling pathway plays essential roles during development and adult tissue homeostasis. Inappropriate activation of the pathway can result in a variety of malignancies. Protein kinases have emerged as key regulators at multiple steps of the Wnt pathway. In this review, we present a synthesis covering the latest information on how Wnt signaling is regulated by diverse protein kinases. PMID:19623618

  5. A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

    PubMed

    Zeng, Xin; Montoute, Monica; Bee, Tiger W; Lin, Hong; Kallal, Lorena A; Liu, Yan; Agarwal, Pankaj; Wang, Dayuan; Lu, Quinn; Morrow, Dwight; Pope, Andrew J; Wu, Zining

    2016-03-01

    Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential. PMID:26656867

  6. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  7. Clinical implications of β-catenin protein expression in breast cancer

    PubMed Central

    Wang, Ziyi; Zhang, Hao; Hou, Jianxin; Niu, Jianing; Ma, Zhenhai; Zhao, Haidong; Liu, Caigang

    2015-01-01

    The objective of this study was to examine the expression and significance of β-catenin in the diagnosis and prognosis of breast cancer. Overall, 241 patients with histologically confirmed breast cancer who had undergone radical surgery were enrolled in this study. β-catenin protein expression in breast cancer samples was evaluated by immunohistochemistry. β-catenin was expressed in Nuclei/Plasma of the samples from 41 patients. β-catenin protein expression correlated with the histological grade of the tumor (P<0.05) and Ki-67 labeling (P<0.01). Survival analysis showed that β-catenin expression negatively correlated with breast cancer-specific survival. Our results showed prominent expression of β-catenin in breast cancer and strongly implicate the β-catenin in tumor promotion. PMID:26823833

  8. The cytoskeletal protein α-catenin unfurls upon binding to vinculin.

    PubMed

    Rangarajan, Erumbi S; Izard, Tina

    2012-05-25

    Adherens junctions (AJs) are essential for cell-cell contacts, morphogenesis, and the development of all higher eukaryotes. AJs are formed by calcium-dependent homotypic interactions of the ectodomains of single membrane-pass cadherin family receptors. These homotypic interactions in turn promote binding of the intracellular cytoplasmic tail domains of cadherin receptors with β-catenin, a multifunctional protein that plays roles in both transcription and AJs. The cadherin receptor-β-catenin complex binds to the cytoskeletal protein α-catenin, which is essential for both the formation and the stabilization of these junctions. Precisely how α-catenin contributes to the formation and stabilization of AJs is hotly debated, although the latter is thought to involve its interactions with the cytoskeletal protein vinculin. Here we report the crystal structure of the vinculin binding domain (VBD) of α-catenin in complex with the vinculin head domain (Vh1). This structure reveals that α-catenin is in a unique unfurled mode allowing dimer formation when bound to vinculin. Finally, binding studies suggest that vinculin must be in an activated state to bind to α-catenin and that this interaction is stabilized by the formation of a ternary α-catenin-vinculin-F-actin complex, which can be formed via the F-actin binding domain of either protein. We propose a feed-forward model whereby α-catenin-vinculin interactions promote their binding to the actin cytoskeleton to stabilize AJs. PMID:22493458

  9. HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

    PubMed Central

    Yu, Nancy; Kakunda, Michael; Pham, Victoria; Lill, Jennie R.; Du, Pan; Wongchenko, Matthew; Yan, Yibing; Firestein, Ron

    2015-01-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study. PMID:25645927

  10. Inhibition of Cyclic Adenosine Monophosphate (cAMP)-response Element-binding Protein (CREB)-binding Protein (CBP)/β-Catenin Reduces Liver Fibrosis in Mice.

    PubMed

    Osawa, Yosuke; Oboki, Keisuke; Imamura, Jun; Kojika, Ekumi; Hayashi, Yukiko; Hishima, Tsunekazu; Saibara, Toshiji; Shibasaki, Futoshi; Kohara, Michinori; Kimura, Kiminori

    2015-11-01

    Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP)/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4)- or bile duct ligation (BDL)-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs) and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80(+) CD11b(+) and Ly6C(low) CD11b(+) macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP)-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis. PMID:26870800

  11. Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

    PubMed Central

    Kong, Yuhan; Zhang, Hongyu; Chen, Xian; Zhang, Wenwen; Zhao, Chen; Wang, Ning; Wu, Ningning; He, Yunfeng; Nan, Guoxin; Zhang, Hongmei; Wen, Sheng; Deng, Fang; Liao, Zhan; Wu, Di; Zhang, Junhui; Qin, Xinyue; Haydon, Rex C.; Luu, Hue H.; He, Tong-Chuan; Zhou, Lan

    2014-01-01

    Background and Aims Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3β may play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling. PMID:24335169

  12. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed

    Alman, B A; Li, C; Pajerski, M E; Diaz-Cano, S; Wolfe, H J

    1997-08-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  13. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed Central

    Alman, B. A.; Li, C.; Pajerski, M. E.; Diaz-Cano, S.; Wolfe, H. J.

    1997-01-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  14. Homeodomain-interacting protein kinase 2 (HIPK2) targets {beta}-catenin for phosphorylation and proteasomal degradation

    SciTech Connect

    Kim, Eun-A; Kim, Ji Eon; Sung, Ki Sa; Choi, Dong Wook; Lee, Byeong Jae; Choi, Cheol Yong

    2010-04-16

    The regulation of intracellular {beta}-catenin levels is central in the Wnt/{beta}-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/{beta}-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of {beta}-catenin and results in the accumulation of {beta}-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of {beta}-catenin. HIPK2 phosphorylates {beta}-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of {beta}-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of {beta}-catenin, thereby potentiated {beta}-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of {beta}-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of {beta}-catenin through its phosphorylation and proteasomal degradation.

  15. Positive cross talk between protein kinase D and β-catenin in intestinal epithelial cells: impact on β-catenin nuclear localization and phosphorylation at Ser552.

    PubMed

    Wang, Jia; Han, Liang; Sinnett-Smith, James; Han, Li-Li; Stevens, Jan V; Rozengurt, Nora; Young, Steven H; Rozengurt, Enrique

    2016-04-01

    Given the fundamental role of β-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with β-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous β-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation (P< 0.01), peaked at 4 h (P< 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in β-catenin-regulated genes and phosphorylation at Ser(552) in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in β-catenin nuclear localization and phosphorylation at Ser(552) in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and β-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of β-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and β-catenin in intestinal epithelial cells, both in vitro and in vivo. PMID:26739494

  16. Protein kinase A activation enhances β-catenin transcriptional activity through nuclear localization to PML bodies.

    PubMed

    Zhang, Mei; Mahoney, Emilia; Zuo, Tao; Manchanda, Parmeet K; Davuluri, Ramana V; Kirschner, Lawrence S

    2014-01-01

    The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling. PMID:25299576

  17. 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.

  18. Chromatin protein HMGB2 regulates articular cartilage surface maintenance via β-catenin pathway

    PubMed Central

    Taniguchi, Noboru; Caramés, Beatriz; Kawakami, Yasuhiko; Amendt, Brad A.; Komiya, Setsuro; Lotz, Martin

    2009-01-01

    The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis. Mechanisms that regulate the unique phenotype of SZ chondrocytes and maintain SZ integrity are unknown. We recently demonstrated that expression of the chromatin protein high mobility group box (HMGB) protein 2 is restricted to the SZ in articular cartilage suggesting a transcriptional regulation involving HMGB2 in SZ. Here, we show that an interaction between HMGB2 and the Wnt/β-catenin pathway regulates the maintenance of the SZ. We found that the Wnt/β-catenin pathway is active specifically in the SZ in normal mouse knee joints and colocalizes with HMGB2. Both Wnt signaling and HMGB2 expression decrease with aging in mouse joints. Our molecular studies show that HMGB2 enhances the binding of Lef-1 to its target sequence and potentiates transcriptional activation of the Lef-1-β-catenin complex. The HMG domain within HMGB2 is crucial for interaction with Lef-1, suggesting that both HMGB2 and HMGB1 may be involved in this function. Furthermore, conditional deletion of β-catenin in cultured mouse chondrocytes induced apoptosis. These findings define a pathway where protein interactions of HMGB2 and Lef-1 enhance Wnt signaling and promote SZ chondrocyte survival. Loss of the HMGB2-Wnt signaling interaction is a new mechanism in aging-related cartilage pathology. PMID:19805379

  19. Role of β-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

    PubMed

    Zemans, Rachel L; McClendon, Jazalle; Aschner, Yael; Briones, Natalie; Young, Scott K; Lau, Lester F; Kahn, Michael; Downey, Gregory P

    2013-03-15

    Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that β-catenin signaling promotes epithelial repair after inflammatory injury, but the β-catenin target genes that mediate this effect are unknown. Herein, we examined which β-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of β-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the β-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that β-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

  20. Structure-Based Design of 1,4-Dibenzoylpiperazines as β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction Inhibitors.

    PubMed

    Wisniewski, John A; Yin, Jinya; Teuscher, Kevin B; Zhang, Min; Ji, Haitao

    2016-05-12

    A small-molecule inhibitor with a 1,4-dibenzoylpiperazine scaffold was designed to match the critical binding elements in the β-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction interface. Inhibitor optimization led to a potent inhibitor that can disrupt the β-catenin/BCL9 interaction and exhibit 98-fold selectivity over the β-catenin/cadherin interaction. The binding mode of new inhibitors was characterized by structure-activity relationships and site-directed mutagenesis studies. Cell-based studies demonstrated that this series of inhibitors can selectively suppress canonical Wnt signaling and inhibit growth of Wnt/β-catenin-dependent cancer cells.

  1. A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin

    PubMed Central

    Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E.; Selfors, Laura M.; White, Anne; Zervantonakis, Ioannis K.; Singh, Pragya; Dhakal, Sabin; Harper, J. Wade; Brugge, Joan S.

    2016-01-01

    Cell-cell adhesion is central to morphogenesis and maintenance of epithelial cell state. We previously identified 27 candidate cell-cell adhesion regulatory proteins (CCARPs) whose down-regulation disrupts epithelial cell-cell adhesion during collective migration. Using a protein interaction mapping strategy, we found that 18 CCARPs link to core components of adherens junctions or desmosomes. We further mapped linkages between the CCARPs and other known cell-cell adhesion proteins, including hits from recent screens uncovering novel components of E-cadherin adhesions. Mechanistic studies of one novel CCARP which links to multiple cell-cell adhesion proteins, the phosphatase DUSP23, revealed that it promotes dephosphorylation of β-catenin at Tyr 142 and enhances the interaction between α- and β-catenin. DUSP23 knockdown specifically diminished adhesion to E-cadherin without altering adhesion to fibronectin matrix proteins. Furthermore, DUSP23 knockdown produced “zipper-like” cell-cell adhesions, caused defects in transmission of polarization cues, and reduced coordination during collective migration. Thus, this study identifies multiple novel connections between proteins that regulate cell-cell interactions and provides evidence for a previously unrecognized role for DUSP23 in regulating E-cadherin adherens junctions through promoting the dephosphorylation of β-catenin. PMID:27255161

  2. Zinc Protoporphyrin Suppresses β-Catenin Protein Expression in Human Cancer Cells: The Potential Involvement of Lysosome-Mediated Degradation

    PubMed Central

    Wang, Shuai; Hannafon, Bethany N.; Lind, Stuart E.; Ding, Wei-Qun

    2015-01-01

    Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both in vitro and in vivo. We have recently demonstrated that ZnPP diminishes β-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP’s suppression of β-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the β-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces β-catenin exportation was rejected by the observation that there was no detectable β-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of β-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of β-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced β-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP’s anticancer action and reveal a potential new strategy for targeting the β-catenin Wnt signaling pathway for cancer therapy. PMID:26000787

  3. Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein.

    PubMed

    Oda, H; Tsukita, S

    1999-04-01

    Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dalpha-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution.

  4. Rational design of selective small-molecule inhibitors for β-catenin/B-cell lymphoma 9 protein-protein interactions.

    PubMed

    Hoggard, Logan R; Zhang, Yongqiang; Zhang, Min; Panic, Vanja; Wisniewski, John A; Ji, Haitao

    2015-09-30

    Selective inhibition of α-helix-mediated protein-protein interactions (PPIs) with small organic molecules provides great potential for the discovery of chemical probes and therapeutic agents. Protein Data Bank data mining using the HippDB database indicated that (1) the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix had few orientations when interacting with the second protein and (2) the hot spot pockets of PPI complexes had different sizes, shapes, and chemical groups when interacting with the same hydrophobic projecting hot spots of α-helix. On the basis of these observations, a small organic molecule, 4'-fluoro-N-phenyl-[1,1'-biphenyl]-3-carboxamide, was designed as a generic scaffold that itself directly mimics the binding mode of the side chains of hydrophobic projecting hot spots at positions i, i + 3, and i + 7 of an α-helix. Convenient decoration of this generic scaffold led to the selective disruption of α-helix-mediated PPIs. A series of small-molecule inhibitors selective for β-catenin/B-cell lymphoma 9 (BCL9) over β-catenin/cadherin PPIs was designed and synthesized. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This new class of inhibitors can selectively disrupt β-catenin/BCL9 over β-catenin/cadherin PPIs, suppress the transactivation of canonical Wnt signaling, downregulate the expression of Wnt target genes, and inhibit the growth of Wnt/β-catenin-dependent cancer cells. PMID:26352795

  5. [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. PMID:27487660

  6. 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.

  7. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    PubMed

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  8. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway

    PubMed Central

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells. PMID:26280346

  9. 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.

  10. The APC tumor suppressor binds to C-terminal binding protein to divert nuclear beta-catenin from TCF.

    PubMed

    Hamada, Fumihiko; Bienz, Mariann

    2004-11-01

    Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector beta-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. Here, we show that a third function of APC in antagonizing beta-catenin involves C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of beta-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. Our evidence indicates that APC is an adaptor between beta-catenin and CtBP and that CtBP lowers the availability of free nuclear beta-catenin for binding to TCF by sequestering APC/beta-catenin complexes. PMID:15525529

  11. Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin.

    PubMed

    Ide, N; Hata, Y; Deguchi, M; Hirao, K; Yao, I; Takai, Y

    1999-03-24

    Synaptic scaffolding molecule (S-SCAM) is a multiple PDZ domain-containing protein, which interacts with neuroligin, a cell adhesion molecule, and the NMDA receptor. In this study, we searched for S-SCAM-interacting proteins and obtained a neuralplakophilin-related armadillo-repeat protein (NPRAP)/delta-catenin. NPRAP/delta-catenin bound to the last PDZ domain of S-SCAM via its carboxyl-terminus in three different cell-free assay systems, was coimmunoprecipitated with S-SCAM from rat crude synaptosomes, and was localized at the excitatory synapses in rat hippocampal neurons. NPRAP/delta-catenin may be implicated in the molecular organization of synaptic junctions through the interaction with S-SCAM.

  12. Rational design of small-molecule inhibitors for β-catenin/T-cell factor protein-protein interactions by bioisostere replacement.

    PubMed

    Yu, Binxun; Huang, Zheng; Zhang, Min; Dillard, Darren R; Ji, Haitao

    2013-03-15

    A new hot spot-based design strategy using bioisostere replacement is reported to rationally design nonpeptidic small-molecule inhibitors for protein-protein interactions. This method is applied to design new potent inhibitors for β-catenin/T-cell factor (Tcf) interactions. Three hot spot regions of Tcf for binding to β-catenin were quantitatively evaluated; the key binding elements around K435 and K508 of β-catenin were derived; a bioisostere library was used to generate new fragments that can match the proposed critical binding elements. The most potent inhibitor, with a molecular weight of 230, has a Kd of 0.531 μM for binding to β-catenin and a Ki of 3.14 μM to completely disrupt β-catenin/Tcf interactions. The binding mode of the designed inhibitors was validated by the site-directed mutagenesis and structure-activity relationship (SAR) studies. This study provides a new approach to design new small-molecule inhibitors that bind to β-catenin and effectively disrupt β-catenin/Tcf interactions specific for canonical Wnt signaling. PMID:23272635

  13. Nucleoporin 62-Like Protein Activates Canonical Wnt Signaling through Facilitating the Nuclear Import of β-Catenin in Zebrafish

    PubMed Central

    Yang, Xiaojie; Gu, Qilin; Lin, Li; Li, Shaoyang; Zhong, Shan

    2015-01-01

    Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, the developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx, and intestine of developing embryos. Activation of the Wnt/β-catenin pathway positively modulates nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. In contrast, knockdown of Nup62l led to ventralized embryos, an impediment to CE movements, and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitation of nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements, and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish. PMID:25605329

  14. Wnt Proteins Induce Dishevelled Phosphorylation via an LRP5/6- Independent Mechanism, Irrespective of Their Ability To Stabilize β-Catenin

    PubMed Central

    González-Sancho, José M.; Brennan, Keith R.; Castelo-Soccio, Leslie A.; Brown, Anthony M. C.

    2004-01-01

    Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of β-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve β-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/β-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with β-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/β-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/β-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to β-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/β-catenin pathway. PMID:15143170

  15. Cdc42-Interacting Protein 4 Represses E-Cadherin Expression by Promoting β-Catenin Translocation to the Nucleus in Murine Renal Tubular Epithelial Cells.

    PubMed

    Xu, Chuou; Zhou, Qiaodan; Liu, Lili; Liu, Ping; Pei, Guangchang; Zeng, Rui; Han, Min; Xu, Gang

    2015-08-14

    Renal fibrosis is an inevitable outcome of end-stage chronic kidney disease. During this process, epithelial cells lose E-cadherin expression. β-Catenin may act as a mediator by accumulation and translocation to the nucleus. Studies have suggested that CIP4, a Cdc42 effector protein, is associated with β-catenin. However, whether CIP4 contributes to E-cadherin loss in epithelial cells by regulating β-catenin translocation is unclear. In this study, we investigated the involvement of CIP4 in β-catenin translocation. Expression of CIP4 was upregulated in renal tissues of 5/6 nephrectomized rats and mainly distributed in renal tubular epithelia. In TGF-β1-treated NRK-52E cells, upregulation of CIP4 expression was accompanied by reduced expression of E-cadherin. CIP4 overexpression promoted the translocation of β-catenin to the nucleus, which was accompanied by reduced expression of E-cadherin even without TGF-β1 stimulation. In contrast, CIP4 depletion by using siRNA inhibited the translocation of β-catenin to the nucleus and reversed the decrease in expression of E-cadherin. The interaction between CIP4 and β-catenin was detected. We also show that β-catenin depletion could restore the expression of E-cadherin that was suppressed by CIP4 overexpression. In conclusion, these results suggest that CIP4 overexpression represses E-cadherin expression by promoting β-catenin translocation to the nucleus.

  16. The structural basis of urea-induced protein unfolding in β-catenin.

    PubMed

    Wang, Chao; Chen, Zhongzhou; Hong, Xia; Ning, Fangkun; Liu, Haolin; Zang, Jianye; Yan, Xiaoxue; Kemp, Jennifer; Musselman, Catherine A; Kutateladze, Tatinna G; Zhao, Rui; Jiang, Chengyu; Zhang, Gongyi

    2014-11-01

    Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation.

  17. Nuclear β-catenin accumulation is associated with increased expression of Nanog protein and predicts poor prognosis of non-small cell lung cancer

    PubMed Central

    2013-01-01

    Background Although the prognostic roles of β-catenin expression in non-small cell lung cancer (NSCLC) have been reported in several immunohistochemical (IHC) studies, the results were not consistent because some studies lack sufficient number of the positive cases or did not evaluate the subcellular localization features of the protein. Method In this study, we have evaluated the expression levels and subcellular localization of β-catenin and Nanog proteins IHC staining in tissue specimens from 309 patients with NSCLC, and explored their association with clinicopathological features and patient outcome. Results We showed that patients with negative expression of membranous beta-catenin had a trend towards shorter survival (p=0.064) than those with positive expression. In contrast to previous studies, we found that increased expression of either cytoplasmic or nuclear β-catenin was strongly associated with poor prognosis and was an independent prognosticator for overall survival (p <0.01). We further found that NSCLC cells frequently exhibited an abundance of nuclear Nanog protein which was significantly correlated with nuclear β-catenin expression (p <0.01) and poor prognosis (p <0.01). Interestingly, immunofluorescent staining results revealed that increased expression of Nanog and nuclear translocation of β-catenin occurred concomitantly in response to epidermal growth factor receptor(EGFR) signaling in A549 and H23 cells. Furthermore, western blot analysis show that nuclear β-catenin rather than cytoplasmic β-catenin expression in the A549 and H23 cells can be enhanced by adding EGF, Nanog expression in the A549 and H23 cells with knockdown of β-catenin can not be obviously enhanced by adding EGF. Conclusion We propose that evaluation of subcellular localization of β-catenin and Nanog expression is of clinical significance for patients with NSCLC. PMID:23648139

  18. Mangiferin exerts antitumor activity in breast cancer cells by regulating matrix metalloproteinases, epithelial to mesenchymal transition, and β-catenin signaling pathway

    SciTech Connect

    Li, Hongzhong; Huang, Jing; Yang, Bing; Xiang, Tingxiu; Yin, Xuedong; Peng, Weiyan; Cheng, Wei; Wan, Jingyuan; Luo, Fuling; Li, Hongyuan; Ren, Guosheng

    2013-10-01

    Although mangiferin which is a naturally occurring glucosylxanthone has exhibited promising anticancer activities, the detailed molecular mechanism of mangiferin on cancers still remains enigmatic. In this study, the anticancer activity of mangiferin was evaluated in breast cancer cell line-based in vitro and in vivo models. We showed that mangiferin treatment resulted in decreased cell viability and suppression of metastatic potential in breast cancer cells. Further mechanistic investigation revealed that mangiferin induced decreased matrix metalloproteinase (MMP)-7 and -9, and reversal of epithelial–mesenchymal transition (EMT). Moreover, it was demonstrated that mangiferin significantly inhibited the activation of β-catenin pathway. Subsequent experiments showed that inhibiting β-catenin pathway might play a central role in mangiferin-induced anticancer activity through modulation of MMP-7 and -9, and EMT. Consistent with these findings in vitro, the antitumor potential was also verified in mangiferin-treated MDA-MB-231 xenograft mice where significantly decreased tumor volume, weight and proliferation, and increased apoptosis were obtained, with lower expression of MMP-7 and -9, vimentin and active β-catenin, and higher expression of E-cadherin. Taken together, our study suggests that mangiferin might be used as an effective chemopreventive agent against breast cancer. - Highlights: • Mangiferin inhibits growth and metastatic potential in breast cancer cells. • Mangiferin down-regulates MMP-7 and -9 in breast cancer cells. • Mangiferin induces the reversal of EMT in metastatic breast cancer cells. • Mangiferin inhibits the activation of β-catenin pathway in breast cancer cells. • Inhibiting β-catenin is responsible for the antitumor activity of mangiferin.

  19. 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.

  20. Matrix Gla protein in tumoral pathology

    PubMed Central

    GHEORGHE, SIMONA ROXANA; CRĂCIUN, ALEXANDRA MĂRIOARA

    2016-01-01

    Matrix Gla protein is a vitamin K-dependent protein secreted by chondrocytes and vascular smooth muscle cells. The presence of matrix Gla protein was reported in arterial and venous walls, lungs, kidney, uterus, heart, tooth cementum and eyes. Several studies identified matrix Gla protein in tumoral pathology. Until recently, it was thought to only have an inhibitory role of physiological and ectopic calcification. New studies demonstrated that it also has a role in physiological and pathological angiogenesis, as well as in tumorigenesis. The aim of this review is to report the latest findings related to the expression and clinical implications of matrix Gla protein in different types of cancer with an emphasis on cerebral tumors. PMID:27547048

  1. Matrix Gla protein in tumoral pathology.

    PubMed

    Gheorghe, Simona Roxana; Crăciun, Alexandra Mărioara

    2016-01-01

    Matrix Gla protein is a vitamin K-dependent protein secreted by chondrocytes and vascular smooth muscle cells. The presence of matrix Gla protein was reported in arterial and venous walls, lungs, kidney, uterus, heart, tooth cementum and eyes. Several studies identified matrix Gla protein in tumoral pathology. Until recently, it was thought to only have an inhibitory role of physiological and ectopic calcification. New studies demonstrated that it also has a role in physiological and pathological angiogenesis, as well as in tumorigenesis. The aim of this review is to report the latest findings related to the expression and clinical implications of matrix Gla protein in different types of cancer with an emphasis on cerebral tumors. PMID:27547048

  2. HER2 and β-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

    PubMed

    Cuello-Carrión, F Darío; Shortrede, Jorge E; Alvarez-Olmedo, Daiana; Cayado-Gutiérrez, Niubys; Castro, Gisela N; Zoppino, Felipe C M; Guerrero, Martín; Martinis, Estefania; Wuilloud, Rodolfo; Gómez, Nidia N; Biaggio, Verónica; Orozco, Javier; Gago, Francisco E; Ciocca, Leonardo A; Fanelli, Mariel A; Ciocca, Daniel R

    2015-02-01

    In human breast cancer, β-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of β-catenin/HER2 has been reported, in the present study we have explored whether β-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed β-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of β-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different β-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in β-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and β-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated β-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

  3. 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.

  4. The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin.

    PubMed

    Wadham, Carol; Gamble, Jennifer R; Vadas, Mathew A; Khew-Goodall, Yeesim

    2003-06-01

    Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified beta-catenin, a component of adherens junctions, as a substrate of Pez by a "substrate trapping" approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of beta-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including beta-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro "wound" assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion. PMID:12808048

  5. Dentin Matrix Proteins in Bone Tissue Engineering.

    PubMed

    Ravindran, Sriram; George, Anne

    2015-01-01

    Dentin and bone are mineralized tissue matrices comprised of collagen fibrils and reinforced with oriented crystalline hydroxyapatite. Although both tissues perform different functionalities, they are assembled and orchestrated by mesenchymal cells that synthesize both collagenous and noncollagenous proteins albeit in different proportions. The dentin matrix proteins (DMPs) have been studied in great detail in recent years due to its inherent calcium binding properties in the extracellular matrix resulting in tissue calcification. Recent studies have shown that these proteins can serve both as intracellular signaling proteins leading to induction of stem cell differentiation and also function as nucleating proteins in the extracellular matrix. These properties make the DMPs attractive candidates for bone and dentin tissue regeneration. This chapter will provide an overview of the DMPs, their functionality and their proven and possible applications with respect to bone tissue engineering.

  6. Ubiquitination of specific mitochondrial matrix proteins.

    PubMed

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. PMID:27157140

  7. Ubiquitination of specific mitochondrial matrix proteins.

    PubMed

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G; Ciechanover, Aaron

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems - at least partially - in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins.

  8. β catenin in health: A review

    PubMed Central

    Prakash, Sharada; Swaminathan, Uma

    2015-01-01

    β catenin belongs to the armadillo family of proteins. It plays a crucial role in developmental and homeostatic processes. Wnts are a family of 19 secreted glycoproteins that transduce multiple signaling cascades, including the canonical Wnt/β catenin pathway, Wnt/Ca2+ pathway and the Wnt/polarity pathway. This is a review on β catenin, Wnt proteins and their secretion, the signaling pathway, the associated factors and the crucial role of β catenin in odontogenesis. PMID:26604501

  9. β catenin in health: A review.

    PubMed

    Prakash, Sharada; Swaminathan, Uma

    2015-01-01

    β catenin belongs to the armadillo family of proteins. It plays a crucial role in developmental and homeostatic processes. Wnts are a family of 19 secreted glycoproteins that transduce multiple signaling cascades, including the canonical Wnt/β catenin pathway, Wnt/Ca(2+) pathway and the Wnt/polarity pathway. This is a review on β catenin, Wnt proteins and their secretion, the signaling pathway, the associated factors and the crucial role of β catenin in odontogenesis. PMID:26604501

  10. The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β.

    PubMed

    Dema, Alessandro; Schröter, Micha Friedemann; Perets, Ekaterina; Skroblin, Philipp; Moutty, Marie Christine; Deàk, Veronika Anita; Birchmeier, Walter; Klussmann, Enno

    2016-09-01

    The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.

  11. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin. PMID:26645068

  12. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

  13. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  14. Aberrant expression of E-cadherin and β-catenin proteins in placenta of bovine embryos derived from somatic cell nuclear transfer.

    PubMed

    Kohan-Ghadr, H R; Smith, L C; Arnold, D R; Murphy, B D; Lefebvre, R C

    2012-01-01

    Abnormal placental development is common in the bovine somatic cell nuclear transfer (SCNT)-derived fetus. In the present study, we characterised the expression of E-cadherin and β-catenin, structural proteins of adherens junctions, in SCNT gestations as a model for impaired placentation. Cotyledonary tissues were separated from pregnant uteri of SCNT (n = 6) and control pregnancies (n = 8) obtained by artificial insemination. Samples were analysed by western blot, quantitative RT-PCR (qRT-PCR) and immunohistochemistry. Bovine trophectoderm cell lines derived from SCNT and control embryos were analysed to compare with the in utero condition. Although no differences in E-cadherin or β-catenin mRNA abundance were observed in fetal tissues between the two groups, proteins encoded by these genes were markedly under-expressed in SCNT trophoblast cells. Immunohistochemistry revealed a different pattern of E-cadherin and total β-catenin localisation in SCNT placentas compared with controls. No difference was observed in subcellular localisation of dephosphorylated active-β-catenin protein in SCNT tissues compared with controls. However, qRT-PCR confirmed that the wingless (WNT)/β-catenin signalling pathway target genes CCND1, CLDN1 and MSX1 were downregulated in SCNT placentas. No differences were detected between two groups of bovine trophectoderm cell lines. Our results suggest that impaired expression of E-cadherin and β-catenin proteins, along with defective β-catenin signalling during embryo attachment, specifically during placentation, is a molecular mechanism explaining insufficient placentation in the bovine SCNT-derived fetus.

  15. Regulation of Ribosomal S6 Protein Kinase-p90rsk, Glycogen Synthase Kinase 3, and β-Catenin in Early Xenopus Development

    PubMed Central

    Torres, Monica A.; Eldar-Finkelman, Hagit; Krebs, Edwin G.; Moon, Randall T.

    1999-01-01

    β-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of β-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and β-catenin. Searching for kinase activities able to phosphorylate the conserved, inhibitory-regulatory GSK-3 residue serine 9, we found p90rsk to be a potential upstream regulator of GSK-3. Overexpression of p90rsk in Xenopus embryos leads to increased steady-state levels of total β-catenin but not of the free soluble protein. Instead, p90rsk overexpression increases the levels of β-catenin in a cell fraction containing membrane-associated cadherins. Consistent with the lack of elevation of free β-catenin levels, ectopic p90rsk was unable to rescue dorsal cell fate in embryos ventralized by UV irradiation. We show that p90rsk is a downstream target of fibroblast growth factor (FGF) signaling during early Xenopus development, since ectopic FGF signaling activates both endogenous and overexpressed p90rsk. Moreover, overexpression of a dominant negative FGF receptor, which blocks endogenous FGF signaling, leads to decreased p90rsk kinase activity. Finally, we report that FGF inhibits endogenous GSK-3 activity in Xenopus embryos. We hypothesize that FGF and p90rsk play heretofore unsuspected roles in modulating GSK-3 and β-catenin. PMID:9891076

  16. Localization of peroxisomal matrix proteins by photobleaching

    SciTech Connect

    Buch, Charlotta; Hunt, Mary C.; Alexson, Stefan E.H.; Hallberg, Einar

    2009-10-16

    The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

  17. Localization of peroxisomal matrix proteins by photobleaching.

    PubMed

    Buch, Charlotta; Hunt, Mary C; Alexson, Stefan E H; Hallberg, Einar

    2009-10-16

    The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

  18. A Simple Method to Assess Abundance of the β-Catenin Signaling Pool in Cells.

    PubMed

    Flozak, Annette S; Lam, Anna P; Gottardi, Cara J

    2016-01-01

    β-catenin (CTNNB1) is a dual-function cell-cell adhesion/transcriptional co-activator protein and an essential transducer of canonical Wnt signals. Although a number of established techniques and reagents are available to quantify the nuclear signaling activity of β-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of β-catenin, and generation of N-terminally hypophosphorylated β-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of β-catenin outside of the cadherin complex appears universally required for β-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of β-catenin in cells, using a GST-tagged form of ICAT (Inhibitor of β-Catenin and Tcf) as an affinity matrix. This method is more sensitive and quantitative than immunofluorescence and may be useful in studies that implicate TCF-independent signaling events. PMID:27590151

  19. {beta}-Catenin/LEF1 activated enamelin expression in ameloblast-like cells

    SciTech Connect

    Tian, Hua; Lv, Ping; Ma, Kangtao; Zhou, Chunyan; Gao, Xuejun

    2010-07-30

    Research highlights: {yields} {beta}-Catenin/LEF1 complex could activate enamelin gene transcription. {yields} {beta}-Catenin/LEF1 can directly bind to enamelin 5' regulatory region. {yields} Wnt/{beta}-catenin signaling can upregulate enamelin expression in ameloblast-like cells. -- Abstract: Enamelin is an ameloblast-specific matrix protein believed to play essential roles in enamel formation. However, mechanisms of enamelin transcription regulation are not clear. {beta}-Catenin/LEF1 is a key transcriptional complex involved in tooth development. In this study, the role of {beta}-catenin/LEF1 in enamelin expression was investigated. The 5'-flanking region of the mouse enamelin gene was analyzed and cloned. Co-transfection analysis and mutation assays revealed that two conserved LEF1 responsive elements located at -1002 and -597 bp upstream of the enamelin translation initiation site could augment transcriptional activity of the enamelin. The interaction between the enamelin elements and {beta}-catenin/LEF1 was further confirmed by electrophoresis mobility shift assays and chromatin immunoprecipitation assays. In addition, LiCl treatment induced nuclear translocation of {beta}-catenin and elevated endogenous enamelin expression in mouse ameloblast-like cells. The results suggested that Wnt/{beta}-catenin signaling could function in enamelin gene expression by direct interaction through two conserved LEF1 responsive elements on the enamelin gene in ameloblast-like cells.

  20. Wnt-dependent beta-catenin signaling is activated after unilateral ureteral obstruction, and recombinant secreted frizzled-related protein 4 alters the progression of renal fibrosis.

    PubMed

    Surendran, Kameswaran; Schiavi, Susan; Hruska, Keith A

    2005-08-01

    beta-Catenin functions as a transducer of Wnt signals to the nucleus, where it interacts with the T cell factor (TCF) family of DNA binding proteins to regulate gene expression. On the basis of the genes regulated by beta-catenin and TCF in various biologic settings, two predicted functions of beta-catenin/TCF-dependent transcription are to mediate the loss of epithelial polarity and to promote fibroblast activities, such as the increased synthesis of fibronectin during chronic renal disease. These predictions were tested by determination of the expression and function of an inhibitor of Wnt signaling, secreted frizzled-related protein 4 (sFRP4), during renal tubular epithelial injury initiated by unilateral ureteral obstruction (UUO). Despite increased sFRP4 gene expression in perivascular regions of injured kidneys, total sFRP4 protein levels decreased after injury. The decreased sFRP4 protein levels after UUO accompanied increased Wnt-dependent beta-catenin signaling in tubular epithelial and interstitial cells, along with increased expression of markers of fibrosis. Administration of recombinant sFRP4 protein caused a reduction in tubular epithelial beta-catenin signaling and suppressed the progression of renal fibrosis, as evidenced by a partial maintenance of E-cadherin mRNA expression and a reduction in the amount of fibronectin and alpha-smooth muscle actin proteins. Furthermore, recombinant sFRP4 reduced the number of myofibroblasts, a central mediator of fibrosis. It is concluded that beta-catenin signaling is activated in tubular epithelial and interstitial cells after renal injury, and recombinant sFRP4 can interfere with epithelial de-differentiation and with fibroblast differentiation and function during progression of renal fibrosis.

  1. Beyond β-catenin: prospects for a larger catenin network in the nucleus.

    PubMed

    McCrea, Pierre D; Gottardi, Cara J

    2016-01-01

    β-catenin is widely regarded as the primary transducer of canonical WNT signals to the nucleus. In most vertebrates, there are eight additional catenins that are structurally related to β-catenin, and three α-catenin genes encoding actin-binding proteins that are structurally related to vinculin. Although these catenins were initially identified in association with cadherins at cell-cell junctions, more recent evidence suggests that the majority of catenins also localize to the nucleus and regulate gene expression. Moreover, the number of catenins reported to be responsive to canonical WNT signals is increasing. Here, we posit that multiple catenins form a functional network in the nucleus, possibly engaging in conserved protein-protein interactions that are currently better characterized in the context of actin-based cell junctions. PMID:26580716

  2. G protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-catenin signaling and are essential for head formation in Xenopus.

    PubMed

    Miyagi, Asuka; Negishi, Takefumi; Yamamoto, Takamasa S; Ueno, Naoto

    2015-11-01

    Patterning of the vertebrate anterior-posterior axis is regulated by the coordinated action of growth factors whose effects can be further modulated by upstream and downstream mediators and the cross-talk of different intracellular pathways. In particular, the inhibition of the Wnt/β-catenin signaling pathway by various factors is critically required for anterior specification. Here, we report that Flop1 and Flop2 (Flop1/2), G protein-coupled receptors related to Gpr4, contribute to the regulation of head formation by inhibiting Wnt/β-catenin signaling in Xenopus embryos. Using whole-mount in situ hybridization, we showed that flop1 and flop2 mRNAs were expressed in the neural ectoderm during early gastrulation. Both the overexpression and knockdown of Flop1/2 resulted in altered embryonic head phenotypes, while the overexpression of either Flop1/2 or the small GTPase RhoA in the absence of bone morphogenetic protein (BMP) signaling resulted in ectopic head induction. Examination of the Flops' function in Xenopus embryo animal cap cells showed that they inhibited Wnt/β-catenin signaling by promoting β-catenin degradation through both RhoA-dependent and -independent pathways in a cell-autonomous manner. These results suggest that Flop1 and Flop2 are essential regulators of Xenopus head formation that act as novel inhibitory components of the Wnt/β-catenin signaling pathway.

  3. Extracellular matrix proteins involved in pseudoislets formation.

    PubMed

    Maillard, Elisa; Sencier, Marie-Christine; Langlois, A; Bietiger, William; Krafft, Mp; Pinget, Michel; Sigrist, Séverine

    2009-01-01

    Extracellular matrix proteins are known to mediate, through integrins, cell adhesion and are involved in a number of cellular processes, including insulin expression and secretion in pancreatic islets. We investigated whether expression of some extracellular matrix proteins were implied in islets-like structure formation, named pseudoislets. For this purpose, we cultured the β-cell line, RINm5F, during 1, 3, 5 and 7 days of culture on treated or untreated culture plate to form adherent cells or pseudoislets and analysed insulin, collagen IV, fibronectin, laminin 5 and β1-integrin expression. We observed that insulin expression and secretion were increased during pseudoislets formation. Moreover, we showed by immunohistochemistry an aggregation of insulin secreting cells in the centre of the pseudoislets. Peripheral β-cells of pseudoislets did not express insulin after 7 days of culture. RT-PCR and immunohistochemistry studies showed a transient expression of type IV collagen in pseudoislets for the first 3 days of culture. Study of fibronectin expression indicated that adherent cells expressed more fibronectin than pseudoislets. In contrast, laminin 5 was more expressed in pseudoislets than in adherent cells. Finally, expression of β1-integrin was increased in pseudoislets as compared to adherent cells. In conclusion, laminin 5 and collagen IV might be implicated in pseudoislets formation whereas fibronectin might be involved in cell adhesion. These data suggested that extracellular matrix proteins may enhance the function of pseudoislets.

  4. All Trans-Retinoic Acid Mediates MED28/HMG Box-Containing Protein 1 (HBP1)/β-Catenin Signaling in Human Colorectal Cancer Cells.

    PubMed

    Lee, Ming-Fen; Hsieh, Nien-Tsu; Huang, Chun-Yin; Li, Chun-I

    2016-08-01

    Vitamin A is required for normal body function, including vision, epithelial integrity, growth, and differentiation. All trans-retinoic acid (ATRA), a family member of vitamin A, has been explored in treating acute promyelocytic leukemia and other types of cancer. Dysregulated Wnt/β-catenin signaling and disrupted cadherin-catenin complex often contribute to colorectal malignancy. MED28, a mammalian Mediator subunit, is found highly expressed in breast and colorectal cancers. Our laboratory has also reported that MED28 regulates cell growth, migration, and invasion in human breast cancer cells. In the current study we investigated the effect of ATRA on MED28 and Wnt/β-catenin signaling in colorectal cancer. HCT116, HT29, SW480, and SW620, four human colorectal cancer cell lines representing different stages of carcinogenesis and harboring critical genetic changes, were employed. Our data indicated that regardless of genetic variations among these cells, suppression of MED28 reduced the expression of cyclin D1, c-Myc, and nuclear β-catenin, but increased the expression of E-cadherin and HMG box-containing protein 1 (HBP1) where HBP1 has been described as a negative regulator of the Wnt/β-catenin signaling. The reporter activity of an HBP1 promoter increased upon MED28 knockdown, but decreased upon MED28 overexpression. ATRA reduced the expression of MED28 and mimicked the effect of MED28 suppression in down-regulating Wnt/β-catenin signaling. Taken together, ATRA can reverse the suppressive effect of MED28 on HBP1 and E-cadherin and inactivate the Wnt/β-catenin pathway in colorectal cancer, suggesting a protective effect of ATRA against colorectal cancer. J. Cell. Physiol. 231: 1796-1803, 2016. © 2015 Wiley Periodicals, Inc.

  5. All Trans-Retinoic Acid Mediates MED28/HMG Box-Containing Protein 1 (HBP1)/β-Catenin Signaling in Human Colorectal Cancer Cells.

    PubMed

    Lee, Ming-Fen; Hsieh, Nien-Tsu; Huang, Chun-Yin; Li, Chun-I

    2016-08-01

    Vitamin A is required for normal body function, including vision, epithelial integrity, growth, and differentiation. All trans-retinoic acid (ATRA), a family member of vitamin A, has been explored in treating acute promyelocytic leukemia and other types of cancer. Dysregulated Wnt/β-catenin signaling and disrupted cadherin-catenin complex often contribute to colorectal malignancy. MED28, a mammalian Mediator subunit, is found highly expressed in breast and colorectal cancers. Our laboratory has also reported that MED28 regulates cell growth, migration, and invasion in human breast cancer cells. In the current study we investigated the effect of ATRA on MED28 and Wnt/β-catenin signaling in colorectal cancer. HCT116, HT29, SW480, and SW620, four human colorectal cancer cell lines representing different stages of carcinogenesis and harboring critical genetic changes, were employed. Our data indicated that regardless of genetic variations among these cells, suppression of MED28 reduced the expression of cyclin D1, c-Myc, and nuclear β-catenin, but increased the expression of E-cadherin and HMG box-containing protein 1 (HBP1) where HBP1 has been described as a negative regulator of the Wnt/β-catenin signaling. The reporter activity of an HBP1 promoter increased upon MED28 knockdown, but decreased upon MED28 overexpression. ATRA reduced the expression of MED28 and mimicked the effect of MED28 suppression in down-regulating Wnt/β-catenin signaling. Taken together, ATRA can reverse the suppressive effect of MED28 on HBP1 and E-cadherin and inactivate the Wnt/β-catenin pathway in colorectal cancer, suggesting a protective effect of ATRA against colorectal cancer. J. Cell. Physiol. 231: 1796-1803, 2016. © 2015 Wiley Periodicals, Inc. PMID:26660958

  6. Nicotine reduces the levels of surfactant proteins A and D via Wnt/β-catenin and PKC signaling in human airway epithelial cells.

    PubMed

    Zou, Weifeng; Liu, Sha; Hu, Jinxing; Sheng, Qing; He, Fang; Li, Bing; Ran, Pixin

    2016-01-15

    A deficiency of surfactant proteins A and D has been proposed as a mechanism in airway remodeling, which is one characteristic of chronic obstructive pulmonary disease (COPD). We recently showed that in vitro nicotine exposure induces Wnt3a/β-catenin activation, which is a pathway that has also been implicated in altering levels of SP-A and SP-D. Nicotine induced activation of protein kinase C(PKC), and the involvement of PKC in mediating Wnt signaling has been demonstrated previously. The main aim of this study was to investigate whether human bronchial epithelial cells reduce levels of SP-A and SP-D in vitro following nicotine stimulation via the Wnt3a/β-catenin and PKC signaling pathway. We showed that nicotine activated the Wnt3a/β-catenin and PKC signaling pathway, and this activation was accompanied by a decrease in SP-A and SP-D expression. Knockdown of Wnt3a with small interfering RNA (siRNA) prevented translocation of β-catenin into the nucleus and reduction levels of SP-A and SP-D. Furthermore, a PKC inhibitor partially prevented these effects,which suggests in HBECs, Wnt3a/β-catenin and PKC pathways interact during nicotine-reduced levels of SP-A and SP-D. These results suggest that HBECs reduce the levels of surfactant proteins A and D in vitro via the Wnt3a/β-catenin and PKC signaling pathway upon nicotine stimulation.

  7. Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin.

    PubMed

    Bonnet, Nicolas; Conway, Simon J; Ferrari, Serge L

    2012-09-11

    Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn(-/-) and Postn(+/+) mice to intermittent PTH. Compared with Postn(+/+), Postn(-/-) mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn(-/-) mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-β-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn(-/-) mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVβ3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn(-/-) mice. In addition, primary osteoblasts from Postn(-/-) mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn(-/-) osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-β-catenin signaling, and osteoblast differentiation.

  8. Extracellular Matrix Proteins in Hemostasis and Thrombosis

    PubMed Central

    Bergmeier, Wolfgang; Hynes, Richard O.

    2012-01-01

    The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets. PMID:21937733

  9. Matrix Gla protein inhibition of tooth mineralization.

    PubMed

    Kaipatur, N R; Murshed, M; McKee, M D

    2008-09-01

    Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice, using an osteoblast/odontoblast-specific 2.3-kb Col1a1 promoter. Mandibles were analyzed by radiography, micro-computed tomography, light microscopy, histomorphometry, and transmission electron microscopy. While bone and tooth ECMs were established in the Col1a1-Mgp mice, extensive hypomineralization was observed, with values of unmineralized ECM from four- to eight-fold higher in dentin and alveolar bone when compared with that in wild-type tissues. Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors. PMID:18719210

  10. Identification of novel NPRAP/δ-catenin-interacting proteins and the direct association of NPRAP with dynamin 2.

    PubMed

    Koutras, Carolina; Lévesque, Georges

    2011-01-01

    Neural plakophilin-related armadillo protein (NPRAP or δ-catenin) is a neuronal-specific protein that is best known for its interaction with presenilin 1 (PS1). Interestingly, the hemizygous loss of NPRAP is associated with severe mental retardation in cri du chat syndrome (CDCS), and mutations in PS1 cause an aggressive, early-onset form of Alzheimer's disease. Until recently, studies on the function of NPRAP have focused on its ability to modulate dendritic protrusion elaboration through its binding to cell adhesion and scaffolding molecules. However, mounting evidence indicates that NPRAP participates in intracellular signaling and exists in the nucleus, where it modulates gene expression. This apparent bifunctional nature suggests an elaborate neuronal role, but how NPRAP came to participate in such distinct subcellular events remains a mystery. To gain insight into this pathway, we immunoprecipitated NPRAP from human SH SY5Y cells and identified several novel interacting proteins by mass spectrometry. These included neurofilament alpha-internexin, interferon regulatory protein 2 binding factors, and dynamins 1 and 2. We further validated dynamin 2/NPRAP colocalization and direct interaction in vivo, confirming their bona fide partnership. Interestingly, dynamin 2 has established roles in endocytosis and actin assembly, and both of these processes have the potential to interface with the cell adhesion and intracellular signaling processes that involve NPRAP. Our data provide new avenues for approaching NPRAP biology and suggest a broader role for this protein than previously thought. PMID:22022388

  11. Green tea polyphenol, (−)-epigallocatechin-3-gallate, induces toxicity in human skin cancer cells by targeting β-catenin signaling

    SciTech Connect

    Singh, Tripti; Katiyar, Santosh K.

    2013-12-01

    The green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), has been shown to have anti-carcinogenic effects in several skin tumor models, and efforts are continued to investigate the molecular targets responsible for its cytotoxic effects to cancer cells. Our recent observation that β-catenin is upregulated in skin tumors suggested the possibility that the anti-skin carcinogenic effects of EGCG are mediated, at least in part, through its effects on β-catenin signaling. We have found that treatment of the A431 and SCC13 human skin cancer cell lines with EGCG resulted in reduced cell viability and increased cell death and that these cytotoxic effects were associated with inactivation of β-catenin signaling. Evidence of EGCG-induced inactivation of β-catenin included: (i) reduced accumulation of nuclear β-catenin; (ii) enhanced levels of casein kinase1α, reduced phosphorylation of glycogen synthase kinase-3β, and increased phosphorylation of β-catenin on critical serine{sup 45,33/37} residues; and (iii) reduced levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are down-stream targets of β-catenin. Treatment of cells with prostaglandin E2 (PGE{sub 2}) enhanced the accumulation of β-catenin and enhanced β-catenin signaling. Treatment with either EGCG or an EP2 antagonist (AH6809) reduced the PGE{sub 2}-enhanced levels of cAMP, an upstream regulator of β-catenin. Inactivation of β-catenin by EGCG resulted in suppression of cell survival signaling proteins. siRNA knockdown of β-catenin in A431 and SCC13 cells reduced cell viability. Collectively, these data suggest that induction of cytotoxicity in skin cancer cells by EGCG is mediated by targeting of β-catenin signaling and that the β-catenin signaling is upregulated by inflammatory mediators. - Highlights: • EGCG inhibits cancer cell viability through inactivation of β-catenin signaling. • Inactivation of β-catenin involves the downregulation of inflammatory mediators. • EGCG

  12. Aberrant Wnt/β-catenin signaling and elevated expression of stem cell proteins are associated with osteosarcoma side population cells of high tumorigenicity.

    PubMed

    Yi, Xi-Jun; Zhao, Yu-Hua; Qiao, Li-Xiang; Jin, Chun-Lei; Tian, Jing; Li, Qiu-Shi

    2015-10-01

    According to the cancer stem cell theory, the presence of a small sub‑population of cancer cells, termed cancer stem cells (CSCs), have a significant implication on cancer treatment and are responsible for tumor recurrence. Previous studies have reported that alterations in the Wnt/β‑catenin signaling are crucial in the maintenance of CSCs. In the present study, the characteristic features and activation of Wnt/β‑catenin signaling in CSCs from osteosarcoma, an aggressive human bone tumor, were investigated. In total, ~2.1% of the cancer stem‑like side population (SP) cells were identified in the osteosarcoma samples. The results of subsequent western blot and reverse transcription‑quantitative polymerase chain reaction analyses revealed that the protein levels of β‑catenin and cyclin D1 were markedly upregulated in the fluorescence‑activated cell sorted osteosarcoma SP cells. In addition, the elevated expression levels of stem cell proteins, including CD133, nestin Oct‑4, Sox‑2 and Nanog were significantly higher in the SP cells, which contributed to self‑renewal and enhanced the proliferation rate of the SP cells. Furthermore, the SP cells were found to be highly invasive and able to form tumors in vivo. Taken together, these data suggested that the identification of novel anticancer drugs, which suppress the Wnt/β‑catenin signaling and its downstream pathway may assist in eradicating osteosarcoma stem cells.

  13. Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin

    PubMed Central

    Bonnet, Nicolas; Conway, Simon J.; Ferrari, Serge L.

    2012-01-01

    Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn−/− and Postn+/+ mice to intermittent PTH. Compared with Postn+/+, Postn−/− mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn−/− mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-β-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn−/− mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVβ3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn−/− mice. In addition, primary osteoblasts from Postn−/− mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn−/− osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-β-catenin signaling, and osteoblast differentiation. PMID:22927401

  14. NF-κB Has a Direct Role in Inhibiting Bmp- and Wnt-Induced Matrix Protein Expression

    PubMed Central

    Tarapore, Rohinton S; Lim, Jason; Tian, Chen; Pacios, Sandra; Xiao, Wenmei; Reid, Daniel; Guan, Hancheng; Mattos, Marcelo; Yu, Bo; Wang, Cun-Yu; Graves, Dana T

    2015-01-01

    The host response to pathogens through nuclear factor κB (NF-κB) is an essential defense mechanism for eukaryotic organisms. NF-κB-mediated host responses inhibit bone and other connective tissue synthesis and are thought to affect the transcription of matrix proteins through multiple indirect pathways. We demonstrate that inhibiting NF-κB in osteoblasts increases osteocalcin expression in vivo in mice with periodontal disease. Mutating NF-κB binding sites on osteocalcin (OC) or bone sialoprotein (Bsp) promoters rescues the negative impact of NF-κB on their transcription and that NF-κB can inhibit Wnt- and Bmp-induced OC and Bsp transcription, even when protein synthesis is inhibited, indicating a direct effect of NF-κB. This inhibition depends on p65-p50 NF-κB heterodimer formation and deacetylation by HDAC1 but is not affected by the noncanonical NF-κB pathway. Moreover, NF-κB reduces Runx2 and β-catenin binding to OC/Bsp promoters independently of their nuclear localization. Thus, inflammatory signals stimulate the direct interaction of NF-κB with response elements to inhibit binding of β-catenin and Runx2 binding to nearby consensus sites and reduce expression of matrix proteins. This direct mechanism provides a new explanation for the rapid decrease in new bone formation after inflammation-related NF-κB activation. PMID:26179215

  15. E-cadherin and alpha-, beta-, and gamma-catenin protein expression is up-regulated in ovarian carcinoma cells in serous effusions.

    PubMed

    Davidson, B; Berner, A; Nesland, J M; Risberg, B; Berner, H S; Tropè, C G; Kristensen, G B; Bryne, M; Ann Florenes, V

    2000-12-01

    The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and alpha-catenin expression, in both effusions and solid tumours, and for beta-catenin in solid tumours (range p<0. 001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of alpha-, beta-, and gamma-catenin when compared with primary carcinomas (p=0.002-0. 015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and beta-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with

  16. Characterization and chromosomal assignment of a human cDNA encoding a protein related to the murine 102-kDa cadherin-associated protein ([alpha]-catenin)

    SciTech Connect

    Claverie, J.M. ); Hardelin, J.P.; Legouis, R.; Levilliers, J.; Petit, C. ); Bougueleret, L. ); Mattei, M.G. )

    1993-01-01

    We report the characterization of a human cDNA encompassing the complete coding region of a 945-residue putative protein (CAP-R) 80% identical to the recently described murine 102-kDa [alpha]-catenin (CAP102). The CAP-R protein mostly differs from CAP102 by the presence of a 48-residue insert. This insert exhibits similarity with a segment of the type 1 neurofibromatosis gene product. The analysis of a publicly available human [open quote]expressed sequence tag[close quotes] collection revealed the existence of another human cDNA more closely related (89% identical) to CAP 102. This strongly suggests that CAP-R is not the human homologue of the murine 102- kDa [alpha]-catenin but a new closely related gene of the vinculin family. This is further supported by the computed mutation rates falling outside the range observed for mammalian orthologous genes. Using in situ hybridization, the CAP-R gene could be mapped to the pll.l-pl2 region of human chromosome 2 and to the homologous B3-D region of mouse chromosome 6. 32 refs., 4 fig.

  17. The coiled-coil domain containing protein Ccdc136b antagonizes maternal Wnt/β-catenin activity during zebrafish dorsoventral axial patterning.

    PubMed

    Wei, Shi; Shang, Hanqiao; Cao, Yu; Wang, Qiang

    2016-07-20

    The coiled-coil domain containing protein CCDC136 is a putative tumor suppressor and significantly down-regulated in gastric and colorectal cancer tissues. However, little is known about its biological functions during vertebrate embryo development. Zebrafish has two CCDC136 orthologs, ccdc136a and ccdc136b, but only ccdc136b is highly expressed during early embryonic development. In this study, we demonstrate that ccdc136b is required for dorsal-ventral axial patterning in zebrafish embryos. ccdc136b morphants display strongly dorsalized phenotypes. Loss- and gain-of-function experiments in zebrafish embryos and mammalian cells show that Ccdc136b is a crucial negative regulator of the Wnt/β-catenin signaling pathway, and plays a critical role in the establishment of the dorsal-ventral axis. We further find that Ccdc136b interacts with APC, promotes the binding affinity of APC with β-catenin and then facilitates the turnover of β-catenin. These results provide the first evidence that CCDC136 regulates zebrafish dorsal-ventral patterning by antagonizing Wnt/β-catenin signal transduction and suggest a potential mechanism underlying its suppressive activity in carcinogenesis.

  18. Octamer-binding protein 4 affects the cell biology and phenotypic transition of lung cancer cells involving β-catenin/E-cadherin complex degradation.

    PubMed

    Chen, Zhong-Shu; Ling, Dong-Jin; Zhang, Yang-De; Feng, Jian-Xiong; Zhang, Xue-Yu; Shi, Tian-Sheng

    2015-03-01

    Clinical studies have reported evidence for the involvement of octamer‑binding protein 4 (Oct4) in the tumorigenicity and progression of lung cancer; however, the role of Oct4 in lung cancer cell biology in vitro and its mechanism of action remain to be elucidated. Mortality among lung cancer patients is more frequently due to metastasis rather than their primary tumors. Epithelial‑mesenchymal transition (EMT) is a prominent biological event for the induction of epithelial cancer metastasis. The aim of the present study was to investigate whether Oct4 had the capacity to induce lung cancer cell metastasis via the promoting the EMT in vitro. Moreover, the effect of Oct4 on the β‑catenin/E‑cadherin complex, associated with EMT, was examined using immunofluorescence and immunoprecipitation assays as well as western blot analysis. The results demonstrated that Oct4 enhanced cell invasion and adhesion accompanied by the downregulation of epithelial marker cytokeratin, and upregulation of the mesenchymal markers vimentin and N‑cadherin. Furthermore, Oct4 induced EMT of lung cancer cells by promoting β‑catenin/E‑cadherin complex degradation and regulating nuclear localization of β‑catenin. In conclusion, the present study indicated that Oct4 affected the cell biology of lung cancer cells in vitro through promoting lung cancer cell metastasis via EMT; in addition, the results suggested that the association and degradation of the β‑catenin/E‑cadherin complex was regulated by Oct4 during the process of EMT.

  19. Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

    PubMed Central

    Mutze, Kathrin; Vierkotten, Sarah; Milosevic, Jadranka; Eickelberg, Oliver; Königshoff, Melanie

    2015-01-01

    ABSTRACT The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair. PMID:26035385

  20. The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression.

    PubMed

    de Bruin, Ruben G; van der Veer, Eric P; Prins, Jurriën; Lee, Dae Hyun; Dane, Martijn J C; Zhang, Huayu; Roeten, Marko K; Bijkerk, Roel; de Boer, Hetty C; Rabelink, Ton J; van Zonneveld, Anton Jan; van Gils, Janine M

    2016-02-24

    Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

  1. The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression

    PubMed Central

    de Bruin, Ruben G.; van der Veer, Eric P.; Prins, Jurriën; Lee, Dae Hyun; Dane, Martijn J. C.; Zhang, Huayu; Roeten, Marko K.; Bijkerk, Roel; de Boer, Hetty C.; Rabelink, Ton J.; van Zonneveld, Anton Jan; van Gils, Janine M.

    2016-01-01

    Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3′UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity. PMID:26905650

  2. Calmodulin-dependent protein kinase II/cAMP response element-binding protein/Wnt/β-catenin signaling cascade regulates angiotensin II-induced podocyte injury and albuminuria.

    PubMed

    Jiang, Lei; Xu, Lingling; Song, Yuxian; Li, Jianzhong; Mao, Junhua; Zhao, Allan Zijian; He, Weichun; Yang, Junwei; Dai, Chunsun

    2013-08-01

    Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and β-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or β-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/β-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/β-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/β-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases. PMID:23803607

  3. β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells

    PubMed Central

    Taiyab, Aftab; Korol, Anna; Deschamps, Paula A.; West-Mays, Judith A.

    2016-01-01

    Purpose Transforming growth factor-β–induced epithelial–mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-β–induced PCO have not been fully characterized. Here, we focus on examining the role of β-catenin/cyclic AMP response element–binding protein (CREB)-binding protein (CBP) and β-catenin/T-cell factor (TCF)-dependent signaling in regulating cytoskeletal dynamics during TGF-β–induced EMT in lens epithelial explants. Methods Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-β2 in the presence or absence of the β-catenin/CBP interaction inhibitor, ICG-001, or the β-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. Results An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-β, and colocalized with F-actin filaments. Inhibition of β-catenin/CBP interactions, but not β-catenin/TCF interactions, led to a decrease in TGF-β–induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, α-smooth muscle actin (α-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of β-catenin/CBP–dependent signaling also prevented TGF-β–induced downregulation of epithelial cadherin (E-cadherin) in lens explants. Conclusions We show that β-catenin/CBP–dependent signaling regulates fascin, MMP9, and α-SMA expression during TGF-β–induced EMT. We demonstrate that β-catenin/CBP–dependent signaling is crucial for TGF-β–induced EMT in the lens. PMID:27787561

  4. Matrix Gla protein reinforces angiogenic resolution.

    PubMed

    Sharma, Bikram; Albig, Allan R

    2013-01-01

    Matrix Gla Protein (MGP) is an ECM molecule commonly associated with dysfunctions of large blood vessels such as arteriosclerosis and atherosclerosis. However, the exact role of MGP in the microvasculature is not clear. Utilizing a mouse MGP knockout model we found that MGP suppresses angiogenic sprouting from mouse aorta restricts microvascular density in cardiac and skeletal muscle, and is an endogenous inhibitor of tumor angiogenesis. Similarly, morpholino based knockdown of MGP in zebrafish embryos caused a progressive loss of luminal structures in intersegmental vessels, a phenotype reminiscent of Dll4/Notch inhibition. Accordingly, MGP suppressed Notch-dependent Hes-1 promoter activity and expression of Jagged1 mRNA relative to Dll4 mRNA. However, inhibition of BMP but not Notch or VEGF signaling reversed the excessive angiogenic sprouting phenotype of MGP knockout aortic rings suggesting that MGP may normally suppress angiogenic sprouting by blocking BMP signaling. Collectively, these results suggest that MGP is a multi-functional inhibitor of normal and abnormal angiogenesis that may function by coordinating with both Notch and BMP signaling pathways. PMID:23110920

  5. Vascular wall extracellular matrix proteins and vascular diseases

    PubMed Central

    Xu, Junyan; Shi, Guo-Ping

    2014-01-01

    Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension. PMID:25045854

  6. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    PubMed Central

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  7. STAT3 paradoxically stimulates β-catenin expression but inhibits β-catenin function

    PubMed Central

    Ibrahem, Salih; Al-Ghamdi, Saleh; Baloch, Kanwal; Muhammad, Belal; Fadhil, Wakkas; Jackson, Darryl; Nateri, Abdolrahman S; Ilyas, Mohammad

    2014-01-01

    Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and β-catenin (through gene knock-down and dominant-negative TCF4 expression) in nine CRC cell lines. β-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced β-catenin mRNA and protein levels. The reduction in β-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased β-catenin mRNA. This suggests that STAT3 positively regulates β-catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear β-catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and β-catenin individually and in combination. Knock-down of β-catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knock-down of STAT3 and β-catenin had a significantly weaker effect than knock-down of β-catenin alone (P < 0.01). Knock-down of STAT3 and β-catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates β-catenin but β-catenin does not regulate STAT3. The STAT3/β-catenin interaction is complex but may reduce the proliferative activity of β-catenin possibly by taking β-catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and β-catenin are activated compared to those where only one is activated. PMID:25348333

  8. The microtubule-associated protein PRC1 promotes early recurrence of hepatocellular carcinoma in association with the Wnt/β-catenin signalling pathway

    PubMed Central

    Chen, Jianxiang; Rajasekaran, Muthukumar; Xia, Hongping; Zhang, Xiaoqian; Kong, Shik Nie; Sekar, Karthik; Seshachalam, Veerabrahma Pratap; Deivasigamani, Amudha; Goh, Brian Kim Poh; Ooi, London Lucien; Hong, Wanjin; Hui, Kam M

    2016-01-01

    Objectives Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality worldwide. Alterations in microtubule-associated proteins (MAPs) have been observed in HCC. However, the mechanisms underlying these alterations remain poorly understood. Our aim was to study the roles of the MAP protein regulator of cytokinesis 1 (PRC1) in hepatocarcinogenesis and early HCC recurrence. Design PRC1 expression in HCC samples was evaluated by microarray, immunoblotting and immunohistochemistry analysis. Molecular and cellular techniques including siRNA-mediated and lentiviral vector-mediated knockdown were used to elucidate the functions and mechanisms of PRC1. Results PRC1 expression was associated with early HCC recurrence and poor patient outcome. In HCC, PRC1 exerted an oncogenic effect by promoting cancer proliferation, stemness, metastasis and tumourigenesis. We further demonstrated that the expression and distribution of PRC1 is dynamically regulated by Wnt3a signalling. PRC1 knockdown impaired transcription factor (TCF) transcriptional activity, decreased Wnt target expression and reduced nuclear β-catenin levels. Mechanistically, PRC1 interacts with the β-catenin destruction complex, regulates Wnt3a-induced membrane sequestration of this destruction complex, inhibits adenomatous polyposis coli (APC) stability and promotes β-catenin release from the APC complex. In vivo, high PRC1 expression correlated with nuclear β-catenin and Wnt target expression. PRC1 acted as a master regulator of a set of 48 previously identified Wnt-regulated recurrence-associated genes (WRRAGs) in HCC. Thus, PRC1 controlled the expression and function of WRRAGs such as FANCI, SPC25, KIF11 and KIF23 via Wnt signalling. Conclusions We identified PRC1 as a novel Wnt target that functions in a positive feedback loop that reinforces Wnt signalling to promote early HCC recurrence. PMID:26941395

  9. Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein*

    PubMed Central

    Barbolina, Maria V.; Liu, Yiuying; Gurler, Hilal; Kim, Mijung; Kajdacsy-Balla, Andre A.; Rooper, Lisa; Shepard, Jaclyn; Weiss, Michael; Shea, Lonnie D.; Penzes, Peter; Ravosa, Matthew J.; Stack, M. Sharon

    2013-01-01

    Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling. PMID:23152495

  10. A specific domain in alpha-catenin mediates binding to beta-catenin or plakoglobin.

    PubMed

    Huber, O; Krohn, M; Kemler, R

    1997-08-01

    The E-cadherin-catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either beta-catenin or plakoglobin, which both can assemble alpha-catenin into the complex. Recently we have identified an alpha-catenin binding site in beta-catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in alpha-catenin which mediates its interaction with beta-catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of alpha-catenin expressed as recombinant fusion proteins, we found that the N-terminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117-143) which is necessary and sufficient to bind beta-catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two alpha-helices.

  11. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin.

    PubMed

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-30

    β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  12. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  13. Gene evolution and functions of extracellular matrix proteins in teeth

    PubMed Central

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-01-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the “core matrisome” in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  14. Nipah virus matrix protein: expert hacker of cellular machines.

    PubMed

    Watkinson, Ruth E; Lee, Benhur

    2016-08-01

    Nipah virus (NiV, Henipavirus) is a highly lethal emergent zoonotic paramyxovirus responsible for repeated human outbreaks of encephalitis in South East Asia. There are no approved vaccines or treatments, thus improved understanding of NiV biology is imperative. NiV matrix protein recruits a plethora of cellular machinery to scaffold and coordinate virion budding. Intriguingly, matrix also hijacks cellular trafficking and ubiquitination pathways to facilitate transient nuclear localization. While the biological significance of matrix nuclear localization for an otherwise cytoplasmic virus remains enigmatic, the molecular details have begun to be characterized, and are conserved among matrix proteins from divergent paramyxoviruses. Matrix protein appropriation of cellular machinery will be discussed in terms of its early nuclear targeting and later role in virion assembly. PMID:27350027

  15. Nipah virus matrix protein: expert hacker of cellular machines.

    PubMed

    Watkinson, Ruth E; Lee, Benhur

    2016-08-01

    Nipah virus (NiV, Henipavirus) is a highly lethal emergent zoonotic paramyxovirus responsible for repeated human outbreaks of encephalitis in South East Asia. There are no approved vaccines or treatments, thus improved understanding of NiV biology is imperative. NiV matrix protein recruits a plethora of cellular machinery to scaffold and coordinate virion budding. Intriguingly, matrix also hijacks cellular trafficking and ubiquitination pathways to facilitate transient nuclear localization. While the biological significance of matrix nuclear localization for an otherwise cytoplasmic virus remains enigmatic, the molecular details have begun to be characterized, and are conserved among matrix proteins from divergent paramyxoviruses. Matrix protein appropriation of cellular machinery will be discussed in terms of its early nuclear targeting and later role in virion assembly.

  16. Estradiol binding to nuclear matrix protein of pig adrenal cortex

    SciTech Connect

    Ungar, F.; Johnson, S.R.; Johnston, J.A.

    1987-05-01

    Binding of TH-estradiol can be shown in vitro after incubation with purified washed nuclei of sow adrenal cortex or with the insoluble nuclear matrix protein isolated from nuclei. The procedure modified after Berezney and Coffey treated washed nuclei sequentially with 1% Triton-X100, DNase, RNase and 2M NaCl to give an insoluble nuclear matrix protein preparation in which most of the phospholipid, DNA, RNA and protein was removed. Reagents were added to 10 mM Tris buffer containing 1 mM phenylmethyl sulfonyl fluoride, dithiothreitol and 0.2 mM or 5.0 mM MgCl2. Each treatment and washes were centrifuged at 4C. Suspensions of nuclei and nuclear matrix protein were incubated at 4C for 24 hrs. with 0.25 to 3.0 ng of TH-estradiol in 0.5 ml 10 mM Tris buffer with 5 mM MgCl2. Scatchard analysis of binding in duplicate or triplicate tubes with or without excess unlabeled estradiol gave specific binding for sow adrenal nuclei and for nuclear matrix protein. Total binding sites varied between 780 to 1380 fmoles/mg protein. Estradiol binding was not shown in the fetal adrenal matrix nor in mitochondria. Noncompetitive controls included progesterone and pregnenolone. Nuclear matrix protein binding of estradiol may have significance in functional or morphological changes of the adrenal cortex in fetal, neonatal, or pubertal development.

  17. Interaction between Cartilage Oligomeric Matrix Protein and Extracellular Matrix Protein 1 Mediates Endochondral Bone Growth

    PubMed Central

    Kong, Li; Tian, Qingyun; Guo, Fengjin; Mucignat, Maria T.; Perris, Roberto; Sercu, Sandy; Merregaert, Joseph; Di Cesare, Paul E.; Liu, Chuan-ju

    2010-01-01

    In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth. PMID:20138147

  18. Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

    PubMed

    Kuznetsov, Andrey S; Polyansky, Anton A; Fleck, Markus; Volynsky, Pavel E; Efremov, Roman G

    2015-09-01

    The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association. PMID:26575933

  19. Tumour suppressor TRIM33 targets nuclear β-catenin degradation

    PubMed Central

    Xue, Jianfei; Chen, Yaohui; Wu, Yamei; Wang, Zhongyong; Zhou, Aidong; Zhang, Sicong; Lin, Kangyu; Aldape, Kenneth; Majumder, Sadhan; Lu, Zhimin; Huang, Suyun

    2014-01-01

    Aberrant activation of β-catenin in the nucleus has been implicated in a variety of human cancers but the fate of nuclear β-catenin is unknown. Here we demonstrate that tripartite motif-containing protein 33 (TRIM33), acting as an E3 ubiquitin ligase, reduces the abundance of nuclear β-catenin protein. TRIM33-mediated β-catenin is destabilized and is GSK-3β or β-TrCP independent. TRIM33 interacts with and ubiquitylates nuclear β-catenin. Moreover, protein kinase Cδ, which directly phosphorylates β-catenin at Ser715, is required for the TRIM33–β-catenin interaction. The function of TRIM33 in suppressing tumour cell proliferation and brain tumour development depends on TRIM33-promoted β-catenin degradation. In human glioblastoma specimens, endogenous TRIM33 levels are inversely correlated with β-catenin. In summary, our findings identify TRIM33 as a tumour suppressor that can abolish tumour cell proliferation and tumorigenesis by degrading nuclear β-catenin. This work suggests a new therapeutic strategy against human cancers caused by aberrant activation of β-catenin. PMID:25639486

  20. Computational biophysical, biochemical, and evolutionary signature of human R-spondin family proteins, the member of canonical Wnt/β-catenin signaling pathway.

    PubMed

    Sharma, Ashish Ranjan; Chakraborty, Chiranjib; Lee, Sang-Soo; Sharma, Garima; Yoon, Jeong Kyo; George Priya Doss, C; Song, Dong-Keun; Nam, Ju-Suk

    2014-01-01

    In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models.

  1. Recent advances in the study of zebrafish extracellular matrix proteins.

    PubMed

    Jessen, Jason R

    2015-05-01

    The zebrafish extracellular matrix (ECM) is a dynamic and pleomorphic structure consisting of numerous proteins that together regulate a variety of cellular and morphogenetic events beginning as early as gastrulation. The zebrafish genome encodes a similar complement of ECM proteins as found in other vertebrate organisms including glycoproteins, fibrous proteins, proteoglycans, glycosaminoglycans, and interacting or modifying proteins such as integrins and matrix metalloproteinases. As a genetic model system combined with its amenability to high-resolution microscopic imaging, the zebrafish allows interrogation of ECM protein structure and function in both the embryo and adult. Accumulating data have identified important roles for zebrafish ECM proteins in processes as diverse as cell polarity, migration, tissue mechanics, organ laterality, muscle contraction, and regeneration. In this review, I highlight recently published data on these topics that demonstrate how the ECM proteins fibronectin, laminin, and collagen contribute to zebrafish development and adult homeostasis.

  2. Regulation of the potential marker for intestinal cells, Bmi1, by β-catenin and the zinc finger protein KLF4: implications for colon cancer.

    PubMed

    Yu, Tianxin; Chen, Xi; Zhang, Wen; Colon, Deannon; Shi, Jiandang; Napier, Dana; Rychahou, Piotr; Lu, Wange; Lee, Eun Y; Weiss, Heidi L; Evers, B Mark; Liu, Chunming

    2012-02-01

    B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear β-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/β-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.

  3. Low expression of secreted frizzled-related protein 2 and nuclear accumulation of β-catenin in aggressive nonfunctioning pituitary adenoma

    PubMed Central

    WU, YOUTU; BAI, JIWEI; HONG, LINCHUAN; LIU, CHUNHUI; YU, SHENGYUAN; YU, GUOQIANG; ZHANG, YAZHUO

    2016-01-01

    The identification of a specific molecular marker for aggressiveness of nonfunctioning pituitary adenomas (NFPAs) is urgently required in order to guide the clinical diagnosis and treatment of NFPAs. In the present study, low expression of secreted frizzled-related protein 2 (sFRP2) in NFPAs was demonstrated by reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. The results confirmed an abnormal accumulation of free β-catenin in the nuclei of NFPAs, which is the core step for the activation of the Wnt canonical signaling pathway. Furthermore, cyclin D1 and c-Myc, the downstream proteins of the Wnt canonical signaling pathway, were overexpressed in aggressive NFPAs. These findings demonstrated the activation of the Wnt canonical signaling pathway in aggressive NFPAs. In addition, sFRP2 expression was observed to be inversely correlated to the aggressiveness of NFPAs. Therefore, sFRP2 may act as a tumor suppressor through modulation of the cellular cytosolic pool of β-catenin in NFPAs. Furthermore, the expression of sFRP2 may serve as a biomarker for NFPAs aggressiveness and prognosis. PMID:27347125

  4. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    SciTech Connect

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  5. Dielectric relaxation in a protein matrix

    SciTech Connect

    Pierce, D.W.; Boxer, S.G.

    1992-06-25

    The dielectric relaxation of a sperm whale ApoMb-DANCA complex is measured by the fluorescence dynamic Stokes shift method. Emission energy increases with decreasing temperature, suggesting that the relaxation activation energies of the rate-limiting motions either depend on the conformational substrate or different types of protein motions with different frequencies participate in the reaction. Experimental data suggest that there may be relaxations on a scale of <100 ps. 61 refs., 7 figs., 2 tabs.

  6. Cartilage oligomeric matrix protein enhances the vascularization of acellular nerves

    PubMed Central

    Cui, Wei-ling; Qiu, Long-hai; Lian, Jia-yan; Li, Jia-chun; Hu, Jun; Liu, Xiao-lin

    2016-01-01

    Vascularization of acellular nerves has been shown to contribute to nerve bridging. In this study, we used a 10-mm sciatic nerve defect model in rats to determine whether cartilage oligomeric matrix protein enhances the vascularization of injured acellular nerves. The rat nerve defects were treated with acellular nerve grafting (control group) alone or acellular nerve grafting combined with intraperitoneal injection of cartilage oligomeric matrix protein (experimental group). As shown through two-dimensional imaging, the vessels began to invade into the acellular nerve graft from both anastomotic ends at day 7 post-operation, and gradually covered the entire graft at day 21. The vascular density, vascular area, and the velocity of revascularization in the experimental group were all higher than those in the control group. These results indicate that cartilage oligomeric matrix protein enhances the vascularization of acellular nerves. PMID:27127495

  7. Localized decrease of {beta}-catenin contributes to the differentiation of human embryonic stem cells

    SciTech Connect

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-08-08

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, {beta}-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of {beta}-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down {beta}-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of {beta}-catenin contributes to the spatial pattern of differentiation in hESC colonies.

  8. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  9. Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

    PubMed Central

    1995-01-01

    Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin. PMID:7790378

  10. Matrix Gla Protein polymorphisms are associated with coronary artery calcification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix Gla Protein (MGP) is a key regulator of vascular calcification. Genetic variation at the MGP locus could modulate the development of coronary artery calcification (CAC). We examined the cross-sectional association between MGP SNPs [rs1800802 (T-138C), rs1800801 (G-7A),and rs4236 (Ala102Thr)...

  11. Cell surface growth in Escherichia coli: distribution of matrix protein.

    PubMed Central

    Begg, K J

    1978-01-01

    Autoradiography of cell envelope "ghosts" from Escherichia coli was used to demonstrate that newly synthesized molecules of "matrix" protein are inserted at random locations over the entire surface of the outer membrane and that, once inserted, these molecules are not thereafter conserved in any fixed spatial location. Images PMID:355219

  12. Early articular cartilage degeneration in a developmental dislocation of the hip model results from activation of β-catenin.

    PubMed

    Ning, Bo; Sun, Jun; Yuan, Yi; Yao, Jie; Wang, Peng; Ma, Ruixue

    2014-01-01

    Developmental dislocation or dysplasia of the hip (DDH) is one of the most common deformities in children. Osteoarthritis (OA) is the most frequent long-term complication. The molecular mechanism of early articular cartilage degeneration in DDH is still unclear. It is well known that β-catenin plays a crucial role in articular cartilage degeneration. The objective of this study was to verify the relationship between β-catenin and DDH cartilage degeneration. We used a DDH model that was established by modification of swaddling position in newborn Wistar rats. The hips were isolated from the DDH model rats and untreated control group at the age of 2, 4, 6 and 8 weeks. β-Catenin gene and protein were investigated by quantitative (q)RT-PCR and immunohistochemistry. Collagen X and matrix metalloproteinase (MMP)-13, markers of early cartilage degeneration, were assessed by qRT-PCR. Primary chondrocytes were cultured from cartilage of two groups at the age of 8 weeks. Expression of β-catenin, collagen X and MMP-13 was detected. Continued high expression of β-catenin was observed in cartilage from DDH model rats. mRNA and protein expression of β-catenin was significantly increased in primary chondrocytes of the DDH model compared with the control group. Collagen X and MMP-13 expression was higher in the cartilage and chondrocytes from DDH model rats than the control group. Our findings suggest that early cartilage degeneration in DDH may result from activation of β-catenin signaling. PMID:24817933

  13. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  14. Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia

    PubMed Central

    2012-01-01

    Background Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells. Methods In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP) -7, MMP-16, cytokeratin (CK) 8/18, laminin, E-cadherin and beta-catenin was studied. Results In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed. Conclusions Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up. PMID:22863036

  15. Targeting the β-catenin nuclear transport pathway in cancer.

    PubMed

    Jamieson, Cara; Sharma, Manisha; Henderson, Beric R

    2014-08-01

    The nuclear localization of specific proteins is critical for cellular processes such as cell division, and in recent years perturbation of the nuclear transport cycle of key proteins has been linked to cancer. In particular, specific gene mutations can alter nuclear transport of tumor suppressing and oncogenic proteins, leading to cell transformation or cancer progression. This review will focus on one such factor, β-catenin, a key mediator of the canonical wnt signaling pathway. In response to a wnt stimulus or specific gene mutations, β-catenin is stabilized and translocates to the nucleus where it binds TCF/LEF-1 transcription factors to transactivate genes that drive tumor formation. Moreover, the nuclear import and accumulation of β-catenin correlates with clinical tumor grade. Recent evidence suggests that the primary nuclear transport route of β-catenin is independent of the classical Ran/importin import machinery, and that β-catenin directly contacts the nuclear pore complex to self-regulate its own entry into the nucleus. Here we propose that the β-catenin nuclear import pathway may provide an opportunity for identification of specific drug targets and inhibition of β-catenin nuclear function, much like the current screening of drugs that block binding of β-catenin to LEF-1/TCFs. Here we will discuss the diverse mechanisms regulating nuclear localization of β-catenin and their potential as targets for anticancer agent development. PMID:24820952

  16. Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis

    PubMed Central

    Sun, Yao; Weng, Yuteng; Zhang, Chenyang; Liu, Yi; Kang, Chen; Liu, Zhongshuang; Jing, Bo; Zhang, Qi; Wang, Zuolin

    2015-01-01

    Proteoglycans play important roles in regulating osteogenesis. Dentin matrix protein 1 (DMP1) is a highly expressed bone extracellular matrix protein that regulates both bone development and phosphate metabolism. After glycosylation, an N-terminal fragment of DMP1 protein was identified as a new proteoglycan (DMP1-PG) in bone matrix. In vitro investigations showed that Ser89 is the key glycosylation site in mouse DMP1. However, the specific role of DMP1 glycosylation is still not understood. In this study, a mutant DMP1 mouse model was developed in which the glycosylation site S89 was substituted with G89 (S89G-DMP1). The glycosylation level of DMP1 was down-regulated in the bone matrix of S89G-DMP1 mice. Compared with wild type mice, the long bones of S89G-DMP1 mice showed developmental changes, including the speed of bone remodeling and mineralization, the morphology and activities of osteocytes, and activities of both osteoblasts and osteoclasts. These findings indicate that glycosylation of DMP1 is a key posttranslational modification process during development and that DMP1-PG functions as an indispensable proteoglycan in osteogenesis. PMID:26634432

  17. Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis.

    PubMed

    Sun, Yao; Weng, Yuteng; Zhang, Chenyang; Liu, Yi; Kang, Chen; Liu, Zhongshuang; Jing, Bo; Zhang, Qi; Wang, Zuolin

    2015-12-04

    Proteoglycans play important roles in regulating osteogenesis. Dentin matrix protein 1 (DMP1) is a highly expressed bone extracellular matrix protein that regulates both bone development and phosphate metabolism. After glycosylation, an N-terminal fragment of DMP1 protein was identified as a new proteoglycan (DMP1-PG) in bone matrix. In vitro investigations showed that Ser(89) is the key glycosylation site in mouse DMP1. However, the specific role of DMP1 glycosylation is still not understood. In this study, a mutant DMP1 mouse model was developed in which the glycosylation site S(89) was substituted with G(89) (S89G-DMP1). The glycosylation level of DMP1 was down-regulated in the bone matrix of S89G-DMP1 mice. Compared with wild type mice, the long bones of S89G-DMP1 mice showed developmental changes, including the speed of bone remodeling and mineralization, the morphology and activities of osteocytes, and activities of both osteoblasts and osteoclasts. These findings indicate that glycosylation of DMP1 is a key posttranslational modification process during development and that DMP1-PG functions as an indispensable proteoglycan in osteogenesis.

  18. Domain organizations of modular extracellular matrix proteins and their evolution.

    PubMed

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  19. Rad6B is a positive regulator of beta-catenin stabilization.

    PubMed

    Shekhar, Malathy P V; Gerard, Brigitte; Pauley, Robert J; Williams, Bart O; Tait, Larry

    2008-03-15

    Mutations in beta-catenin or other Wnt pathway components that cause beta-catenin accumulation occur rarely in breast cancer. However, there is some evidence of beta-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of beta-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces beta-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, beta-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus-Wnt-1 mammary tumor-derived cells, implicating Rad6B in physiologic regulation of beta-catenin stability and activity. Ubiquitinated beta-catenin was detectable in chromatin immunoprecipitations performed with beta-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of beta-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular beta-catenin but with minimal effects on cell membrane-associated beta-catenin. Measurement of beta-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular beta-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates beta-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of beta-catenin, reveal a positive regulatory feedback loop between Rad6B and beta-catenin and a novel mechanism of beta-catenin stabilization/activation in breast cancer cells.

  20. Threonine 393 of beta-catenin regulates interaction with Axin.

    PubMed

    Wu, Hao; Symes, Karen; Seldin, David C; Dominguez, Isabel

    2009-09-01

    CK2 is a regulatory kinase implicated in embryonic development and in cancer. Among the CK2 substrates is beta-catenin, a protein with dual function in Wnt signaling and cell adhesion. Previously, we reported that CK2 activity is required for beta-catenin stability and we identified threonine (T) 393 as a major CK2 phosphorylation site in beta-catenin. However, it is not known whether phosphorylation at T393 increases beta-catenin stability and if so, what is the mechanism. In this study we investigate the molecular mechanism of beta-catenin stabilization through phosphorylation at T393. We found that pseudophosphorylation of beta-catenin at T393 resulted in a stable activated form of beta-catenin with decreased affinity for Axin in vitro. This phosphomimetic mutant also displayed decreased regulation by Axin in vivo in a bioassay in Xenopus laevis embryos. In contrast, the binding of T393 pseudophosphorylated beta-catenin to E-cadherin was unaffected. Further analysis showed that pseudophosphorylation at T393 did not prevent beta-catenin phosphorylation by GSK3beta. Interestingly, we found that in the presence of pseudophophorylated beta-catenin and another activated form of beta-catenin, the recruitment of GSK3beta to Axin is enhanced. These findings indicate that phosphorylation of T393 by CK2 may affect the stability of beta-catenin through decreased binding to Axin. In addition, the increased recruitment of GSK3beta to the destruction complex in the presence of activated beta-catenin mutants could be a feedback mechanism to suppress overactive Wnt signaling.

  1. Procaine Inhibits Osteo/Odontogenesis through Wnt/β-Catenin Inactivation

    PubMed Central

    Herencia, Carmen; Diaz-Tocados, Juan Miguel; Jurado, Lidia; Montes de Oca, Addy; Rodríguez-Ortiz, Maria Encarnación; Martín-Alonso, Carmen; Martínez-Moreno, Julio M.; Vergara, Noemi; Rodríguez, Mariano; Almadén, Yolanda; Muñoz-Castañeda, Juan R.

    2016-01-01

    Introduction Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. Methods In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. Results Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. Conclusions Our results demonstrated that procaine administration drastically reduced the mineralization and osteo/odontogenesis of bone marrow mesenchymal stem cells inhibiting Wnt/β-catenin pathway through the increase of Gsk3β expression and β-catenin phosphorylation. PMID:27257912

  2. Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Pandit, Kusum; Ben-Yehudah, Ahmi; Chu, Yanxia; Richards, Thomas; Sciurba, Joshua; Myerburg, Michael; Zhang, Yingze; Parwani, Anil V.; Gibson, Kevin F.; Kaminski, Naftali

    2013-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded. PMID:24376648

  3. Interaction of EGFR to δ-catenin leads to δ-catenin phosphorylation and enhances EGFR signaling

    PubMed Central

    He, Yongfeng; Ryu, Taeyong; Shrestha, Nensi; Yuan, Tingting; Kim, Hangun; Shrestha, Hridaya; Cho, Young-Chang; Seo, Young-Woo; Song, Woo Keun; Kim, Kwonseop

    2016-01-01

    Expression of δ-catenin reportedly increases during late stage prostate cancer. Furthermore, it has been demonstrated that expression of EGFR is enhanced in hormone refractory prostate cancer. In this study, we investigated the possible correlation between EGFR and δ-catenin in prostate cancer cells. We found that EGFR interacted with δ-catenin and the interaction decreased in the presence of EGF. We also demonstrated that, on one hand, EGFR phosphorylated δ-catenin in a Src independent manner in the presence of EGF and on the other hand, δ-catenin enhanced protein stability of EGFR and strengthened the EGFR/Erk1/2 signaling pathway. Our findings added a new perspective to the interaction of EGFR to the E-cadherin complex. They also provided novel insights to the roles of δ-catenin in prostate cancer cells. PMID:26883159

  4. Conformational plasticity of the Ebola virus matrix protein

    PubMed Central

    Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

    2014-01-01

    Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. PMID:25159197

  5. Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

    PubMed

    Wu, Siva; Baum, Marc M; Kerwin, James; Guerrero, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-12-01

    Nontypeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen, can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

  6. The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling.

    PubMed

    Borgal, Lori; Habbig, Sandra; Hatzold, Julia; Liebau, Max C; Dafinger, Claudia; Sacarea, Ilinca; Hammerschmidt, Matthias; Benzing, Thomas; Schermer, Bernhard

    2012-07-20

    Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease and represents the most common genetic cause for end-stage renal disease in children and adolescents. It can be caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs). All NPHPs localize to primary cilia, classifying this disease as a "ciliopathy." The primary cilium is a critical regulator of several cell signaling pathways. Cystogenesis in the kidney is thought to involve overactivation of canonical Wnt signaling, which is negatively regulated by the primary cilium and several NPH proteins, although the mechanism remains unclear. Jade-1 has recently been identified as a novel ubiquitin ligase targeting the canonical Wnt downstream effector β-catenin for proteasomal degradation. Here, we identify Jade-1 as a novel component of the NPHP protein complex. Jade-1 colocalizes with NPHP1 at the transition zone of primary cilia and interacts with NPHP4. Furthermore, NPHP4 stabilizes protein levels of Jade-1 and promotes the translocation of Jade-1 to the nucleus. Finally, NPHP4 and Jade-1 additively inhibit canonical Wnt signaling, and this genetic interaction is conserved in zebrafish. The stabilization and nuclear translocation of Jade-1 by NPHP4 enhances the ability of Jade-1 to negatively regulate canonical Wnt signaling. Loss of this repressor function in nephronophthisis might be an important factor promoting Wnt activation and contributing to cyst formation.

  7. Magnesium inhibits Wnt/β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

    PubMed

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M; Madueño, Juan A; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  8. Hyperunstable matrix proteins in the byssus of Mytilus galloprovincialis

    PubMed Central

    Sagert, Jason; Waite, J. Herbert

    2009-01-01

    Summary The marine mussel Mytilus galloprovincialis is tethered to rocks in the intertidal zone by a holdfast known as the byssus. Functioning as a shock absorber, the byssus is composed of threads, the primary molecular components of which are collagen-containing proteins (preCOLs) that largely dictate the higher order self-assembly and mechanical properties of byssal threads. The threads contain additional matrix components that separate and perhaps lubricate the collagenous microfibrils during deformation in tension. In this study, the thread matrix proteins (TMPs), a glycine-, tyrosine- and asparagine-rich protein family, were shown to possess unique repeated sequence motifs, significant transcriptional heterogeneity and were distributed throughout the byssal thread. Deamidation was shown to occur at a significant rate in a recombinant TMP and in the byssal thread as a function of time. Furthermore, charge heterogeneity presumably due to deamidation was observed in TMPs extracted from threads. The TMPs were localized to the preCOL-containing secretory granules in the collagen gland of the foot and are assumed to provide a viscoelastic matrix around the collagenous fibers in byssal threads. PMID:19561212

  9. Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

    PubMed

    Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

    2016-04-01

    High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. PMID:27107012

  10. Thermal stability of matrix protein from Newcastle disease virus.

    PubMed

    Morán, Irene Sánchez; Cuadrado-Castano, Sara; Barroso, Isabel Muñoz; Kostetsky, Eduard Ya; Zhadan, Galina; Gómez, Javier; Shnyrov, Valery L; Villar, Enrique

    2013-10-01

    The thermal stability of the matrix protein (M protein) of Newcastle disease virus (NDV) has been investigated using high-sensitivity differential scanning calorimetry (DSC) at pH 7.4. The thermal folding/unfolding of M protein at this pH value is a reversible process involving a highly cooperative transition between folded and unfolded monomers with a transition temperature (Tm) of 63 °C, an unfolding enthalpy, ΔH(Tm), of 340 kcal mol(-1), and the difference in heat capacity between the native and denatured states of the protein, ΔCp, of 5.1 kcal K(-1) mol(-1). The heat capacity of the native state of the protein is in good agreement with the values calculated using a structure-based parameterization, whereas the calculated values for the hypothetical fully-unfolded state of the protein is higher than those determined experimentally. This difference between the heat capacity of denatured M protein and the heat capacity expected for an unstructured polypeptide of the same sequence, together with the data derived from the heat-induced changes in the steady-state fluorescence of the protein, indicates that the polypeptide chain maintains a significant amount of residual structure after thermal denaturation. PMID:23916643

  11. Role of matrix protein in cytopathogenesis of vesicular stomatitis virus.

    PubMed Central

    Blondel, D; Harmison, G G; Schubert, M

    1990-01-01

    The matrix (M) protein of vesicular stomatitis virus (VSV) plays an important structural role in viral assembly, and it also has a regulatory role in viral transcription. We demonstrate here that the M protein has an additional function. It causes visible cytopathic effects (CPE), as evidenced by the typical rounding of polygonal cells after VSV infection. We have analyzed a temperature-sensitive mutant of the M protein of VSV (tsG33) which is defective in viral assembly and which fails to cause morphological changes of the cells after infection at the nonpermissive temperature (40 degrees C). Interestingly, this defect in viral assembly as well as the CPE were reversible. Microinjection of antisense oligonucleotides which specifically inhibit M protein translation also inhibited the occurrence of CPE. Most importantly, when cells were transfected with a cDNA encoding the temperature-sensitive M protein of tsG33, no CPE was observed at the nonpermissive temperature. However, when these cells were shifted to the permissive temperature (32 degrees C), they rounded up and detached from the dish. These results demonstrate that M protein in the absence of the other viral proteins causes rounding of the cells, probably through a disorganization of the cytoskeleton. The absence of CPE at the nonpermissive temperature is correlated with an abnormal dotted staining pattern of M in these cells, suggesting that the mutant M protein may self-aggregate or associate with membranes rather than interact with cytoskeletal elements. Images PMID:2157054

  12. Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

    PubMed

    Lee, Wendy; Swarup, Sharan; Chen, Joanna; Ishitani, Tohru; Verheyen, Esther M

    2009-01-01

    The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling. PMID:19088090

  13. The molecular elasticity of the extracellular matrix protein tenascin

    NASA Astrophysics Data System (ADS)

    Oberhauser, Andres F.; Marszalek, Piotr E.; Erickson, Harold P.; Fernandez, Julio M.

    1998-05-01

    Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were ~25nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain,. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain,.

  14. [Glycation of extracellular matrix proteins and its role in atherosclerosis].

    PubMed

    Kuzan, Aleksandra; Chwiłkowska, Agnieszka; Kobielarz, Magdalena; Pezowicz, Celina; Gamian, Andrzej

    2012-10-29

    Glycation consists in formation of advanced glycation end-products (AGE) during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins' expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such as pyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  15. Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

    PubMed

    Yazaki, Ikuko; Tsurugaya, Toko; Santella, Luigia; Chun, Jong Tai; Amore, Gabriele; Kusunoki, Shinichiro; Asada, Akiko; Togo, Tatsuru; Akasaka, Koji

    2015-06-01

    Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

  16. Enamel matrix protein derivatives: role in periodontal regeneration

    PubMed Central

    Rathva, Vandana J

    2011-01-01

    The role of regenerative periodontal therapy is the reconstitution of lost periodontal structures, ie, new formation of root cementum, periodontal ligament, and alveolar bone. The outcome of basic research has pointed to the important role of enamel matrix protein derivative (EMD) in periodontal wound healing. Histologic results from animal and human studies have shown that treatment with EMD promotes periodontal regeneration. Moreover, clinical studies have indicated that treatment with EMD positively influences periodontal wound healing in humans. The goal of this paper is to review the existing literature on EMD. PMID:23674918

  17. p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

    PubMed

    Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

    2015-03-01

    The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

  18. Isolation, purification, and partial characterization of Brucella abortus matrix protein.

    PubMed

    Moriyon, I; Berman, D T

    1983-01-01

    Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout. PMID:6401696

  19. Isolation, purification, and partial characterization of Brucella abortus matrix protein.

    PubMed

    Moriyon, I; Berman, D T

    1983-01-01

    Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout.

  20. Expression of genes encoding extracellular matrix proteins: a macroarray study.

    PubMed

    Futyma, Konrad; Miotła, Paweł; Różyńska, Krystyna; Zdunek, Małgorzata; Semczuk, Andrzej; Rechberger, Tomasz; Wojcierowski, Jacek

    2014-12-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

  1. ADAMTS-12 Associates with and Degrades Cartilage Oligomeric Matrix Protein*

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Xu, Ke; Luan, Yi; Ilalov, Kiril; Sehgal, Bantoo; Yu, Shuang; Howell, Ronald D.; Cesare, Paul E. Di

    2006-01-01

    Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis. PMID:16611630

  2. A homologue gene of β-catenin participates in the development of shrimps and immune response to bacteria and viruses.

    PubMed

    Xie, Ya-Kai; Ding, Ding; Wang, Hui-Min; Kang, Cui-Jie

    2015-11-01

    β-Catenin is a multifunctional protein that is involved in many physiological processes, including development, cell proliferation, cell migration, and apoptosis. However, the function of β-Catenin in crustacean is unknown. In this study, the first shrimp homologous gene of β-catenin in Marsupenaeus japonicus (i.e., Mjβ-catenin) was identified and characterized. The full-length of the complementary DNA of Mjβ-catenin is 3130 bp, including a 2463 bp open reading frame that encodes 821 amino acid. Multiple alignment of β-Catenin proteins suggested that the Armadillo/β-Catenin-like repeat domains were conserved. Phylogenetic analysis showed that β-Catenin from shrimp was clustered into one group with invertebrate β-Catenin. The transcription of β-catenin in various development stages of shrimp was detected and persistently increased as the shrimp matured. In adult shrimp, β-catenin was widely distributed in detected tissues and has the relatively high expression level in gills, hemocytes, testes, and ovaries. The transcripts of β-catenin in tissues of adult shrimp were significantly up-regulated at various time points after infecting with Staphylococcus aureus, Vibrio anguillarum, and white-spot syndrome virus. Furthermore, knockdown of β-catenin resulted in impaired bacterial clearance ability and increased virus copy in shrimp in vivo. Therefore, β-Catenin in shrimp participates in the development and immune response of shrimps. PMID:26334791

  3. Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin.

    PubMed

    Kobayashi, Masaki; Kawabata, Keigo; Kusaka-Kikushima, Ayumi; Sugiyama, Yoshinori; Mabuchi, Tomotaka; Takekoshi, Susumu; Miyasaka, Muneo; Ozawa, Akira; Sakai, Shingo

    2016-06-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-β antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-β-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process. PMID:26968261

  4. Immunohistochemical localization of dentin matrix protein 1 in human dentin.

    PubMed

    Orsini, G; Ruggeri, A; Mazzoni, A; Nato, F; Falconi, M; Putignano, A; Di Lenarda, R; Nanci, A; Breschi, L

    2008-01-01

    Dentin matrix protein 1 (DMP1) is a non-collagenous matrix protein with a recognized role in the formation of mineralized tissues such as dentin. The aim of this study was to analyze the distribution of DMP1 in human dentin by means of immunofluorescence and high-resolution immunogold labeling. Fully developed, sound human dentin specimens were submitted to fluorescence labeling and post-embedding immunolabeling techniques with a rabbit polyclonal antihuman DMP1 antibody followed by corresponding fluorochrome-conjugated or gold-conjugated secondary antibodies. Both immunofluorescence and immunogold labeling showed an intense labeling associated with the peritubular dentin. In addition, at the ultrastructural level, there was also a moderate and diffuse immunoreaction over intertubular dentin, and a weak labeling within predentin which increased in density towards the mineralization front. This study suggests that in adult human teeth, like in rodents, DMP1 is prevalently concentrated at the level of peritubular dentin and this feature is preserved also in fully developed-teeth. These data are consistent with what has been observed in rodents and suggest that DMP1 plays a role in maintenance of the dentin tubular space.

  5. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    NASA Technical Reports Server (NTRS)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  6. Extracellular matrix proteins and the dynamics of dentin formation.

    PubMed

    Butler, William T; Brunn, Jan C; Qin, Chunlin; McKee, Marc D

    2002-01-01

    Dentinogenesis involves controlled reactions that result in conversion of unmineralized predentin to dentin when apatite crystals are formed. This process is dynamic: Maturation events occur within predentin beginning at the proximal layer and progressing to the predentin-dentin (PD) border. One type of controlled reaction is the proteolytic processing of dentin sialophosphoprotein (DSPP) to dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), by cleavage of at least three highly conserved peptide bonds. We postulate that this processing event represents an activation step, resulting in release of DPP, which is active in its effects on formation and growth of apatite crystals. Dentin matrix protein 1 (DPM1), present as a processed fragment (57-kD protein) in bone, is seen in dentin on sodium dodecyl sulfate polyacrylamide gel electrophoresis as one intact protein of 150-200 kD. Anti-57-kD antibodies elicit immunoreactivity in bone, dentin, and cellular cementum. In bone, the reactivity is associated with osteocytes and their cell processes. Similarly, dentin shows reactivity in odontoblasts, predentin, and the odontoblast processes. In summary, the processing of large sialic acid-rich proteins into smaller fragments may be an important part of the controlled conversion of predentin to dentin and osteoid to bone.

  7. NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

    PubMed Central

    Brown, Lola A.; Cox, Cassiah; Baptiste, Janae; Summers, Holly; Button, Ryan; Bahlow, Kennedy; Spurrier, Vaughn; Kyser, Jenna; Luttge, Benjamin G.; Kuo, Lillian; Freed, Eric O.; Summers, Michael F.

    2015-01-01

    Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMAQ5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P2 from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P2 cellular signaling system to direct intracellular Gag trafficking during virus assembly. PMID:25941825

  8. A novel miR-34a target, protein kinase D1, stimulates cancer stemness and drug resistance through GSK3/β-catenin signaling in breast cancer

    PubMed Central

    Chang, EunSun; Kang, Hyeok-Gu; Koo, Yoonjin; Lee, Eun Ji; Ko, Je Yeong; Kong, Hyun Kyung; Chun, Kyung-Hee; Park, Jong Hoon

    2016-01-01

    One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3′-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer. PMID:26895471

  9. Promoter swapping between the genes for a novel zinc finger protein and beta-catenin in pleiomorphic adenomas with t(3;8)(p21;q12) translocations.

    PubMed

    Kas, K; Voz, M L; Röijer, E; Aström, A K; Meyen, E; Stenman, G; Van de Ven, W J

    1997-02-01

    Pleiomorphic adenoma of the salivary glands is a benign epithelial tumour occurring primarily in the major and minor salivary glands. It is by far the most common type of salivary gland tumour. Microscopically, pleiomorphic adenomas show a marked histological diversity with epithelial, myoepithelial and mesenchymal components in a variety of patterns. In addition to a cytogenetic subgroup with normal karyotypes, pleiomorphic adenomas are characterized by recurrent chromosome rearrangements, particularly reciprocal translocations, with breakpoints at 8q12, 3p21, and 12q13-15, in that order of frequency. The most common abnormality is a reciprocal t(3;8)(p21;q12). We here demonstrate that the t(3;8)(p21;q12) results in promoter swapping between PLAG1, a novel, developmentally regulated zinc finger gene at 8q12, and the constitutively expressed gene for beta-catenin (CTNNB1), a protein interface functioning in the WG/WNT signalling pathway and specification of cell fate during embryogenesis. Fusions occur in the 5'-non-coding regions of both genes, exchanging regulatory control elements while preserving the coding sequences. Due to the t(3;8)(p21;q12), PLAG1 is activated and expression levels of CTNNB1 are reduced. Activation of PLAG1 was also observed in an adenoma with a variant translocation t(8;15)(q12;q14). Our results indicate that PLAG1 activation due to promoter swapping is a crucial event in salivary gland tumourigenesis.

  10. {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.

  11. Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

    PubMed Central

    Löhr, M.; Trautmann, B.; Göttler, M.; Peters, S.; Zauner, I.; Maillet, B.; Klöppel, G.

    1994-01-01

    Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8286197

  12. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  13. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

    PubMed

    Edginton, Ryan S; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C Peter; Palombo, Francesca

    2016-01-01

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis. PMID:27684584

  14. Characterization of canine platelet adhesion to extracellular matrix proteins.

    PubMed

    Pelagalli, Alessandra; Pero, Maria Elena; Mastellone, Vincenzo; Cestaro, Anna; Signoriello, Simona; Lombardi, Pietro; Avallone, Luigi

    2011-07-01

    Canine platelets have been extensively studied but little is known about specific aspects such as adhesion. Platelet adhesion is a critical step during haemostasis and thrombosis as well as during inflammatory and immunopathogenic responses. The aim of this study was to evaluate the adhesive properties of canine platelets using fibrinogen and collagen as substrates immobilized on plates. Adhesion was monitored for 120 min and the effect of adenosine 5'-diphosphate (ADP) was assayed. The results showed that canine platelets displayed good adhesion activity that was significantly time-dependent. Moreover, ADP was able to enhance platelet adhesion in a dose-dependent manner. The findings aid knowledge of the adhesion process and suggest a specific role of surface platelet receptors in mediating the interaction with extracellular matrix proteins.

  15. Dentin noncollagenous matrix proteins in familial hypophosphatemic rickets.

    PubMed

    Gaucher, Céline; Boukpessi, Tchilalo; Septier, Dominique; Jehan, Frédéric; Rowe, Peter S; Garabédian, Michèle; Goldberg, Michel; Chaussain-Miller, Catherine

    2009-01-01

    Familial hypophosphatemic rickets is transmitted in most cases as an X-linked dominant trait and results from the mutation of the PHEX gene predominantly expressed in osteoblast and odontoblast. Patients with rickets have been reported to display important dentin defects. Our purpose was to explore the structure, composition and distribution of noncollagenous proteins (NCPs) of hypophosphatemic dentin. We collected teeth from 10 hypophosphatemic patients whose mineralization occurred either in a hypophosphatemic environment or in a corrected phosphate and vitamin environment. Teeth were examined by scanning electron microscopy, immunohistochemistry and Western blot analysis. An abnormal distribution (accumulation in interglobular spaces) and cleavage of the NCPs and particularly of matrix extracellular phosphoglycoprotein were observed in deciduous dentin. In contrast, it was close to normal in permanent dentin mineralized under corrected conditions. In conclusion, dentin mineralization in a corrected phosphate and vitamin D environment compensates the adverse effect of PHEX mutation.

  16. Recruitment of β-catenin to N-cadherin is necessary for smooth muscle contraction.

    PubMed

    Wang, Tao; Wang, Ruping; Cleary, Rachel A; Gannon, Olivia J; Tang, Dale D

    2015-04-01

    β-Catenin is a key component that connects transmembrane cadherin with the actin cytoskeleton at the cell-cell interface. However, the role of the β-catenin/cadherin interaction in smooth muscle has not been well characterized. Here stimulation with acetylcholine promoted the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. Knockdown of β-catenin by lentivirus-mediated shRNA attenuated smooth muscle contraction. Nevertheless, myosin light chain phosphorylation at Ser-19 and actin polymerization in response to contractile activation were not reduced by β-catenin knockdown. In addition, the expression of the β-catenin armadillo domain disrupted the recruitment of β-catenin to N-cadherin. Force development, but not myosin light chain phosphorylation and actin polymerization, was reduced by the expression of the β-catenin armadillo domain. Furthermore, actin polymerization and microtubules have been implicated in intracellular trafficking. In this study, the treatment with the inhibitor latrunculin A diminished the interaction of β-catenin with N-cadherin in smooth muscle. In contrast, the exposure of smooth muscle to the microtubule depolymerizer nocodazole did not affect the protein-protein interaction. Together, these findings suggest that smooth muscle contraction is mediated by the recruitment of β-catenin to N-cadherin, which may facilitate intercellular mechanotransduction. The association of β-catenin with N-cadherin is regulated by actin polymerization during contractile activation.

  17. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells. PMID:24635174

  18. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells.

  19. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling.

    PubMed

    Verkaar, Folkert; Blankesteijn, W Matthijs; Smits, Jos F M; Zaman, Guido J R

    2010-04-01

    Wnt/beta-catenin signaling is an important regulator of cell polarity, proliferation, and stem cell maintenance during development and adulthood. Wnt proteins induce the nuclear accumulation of beta-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/beta-catenin signaling has been implicated in a plethora of pathologies and, most notably, underlies initiation and expansion of several cancers. Here, we apply enzyme fragment complementation to measure the nuclear accumulation of beta-catenin. beta-Catenin was tagged with a peptide fragment of beta-galactosidase and transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus. Stimulation of the cells with recombinant Wnt-3a restored beta-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses beta-catenin-driven reporter gene activity downstream of nuclear entry of beta-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce beta-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was identified as an activator of Wnt/beta-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous beta-catenin protein and can induce TCF/LEF-dependent gene transcription. Subsequent biochemical profiling of >200 kinases revealed both isoforms of glycogen synthase kinase 3, as previously unappreciated targets of sotrastaurin. We show that the beta-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/beta-catenin pathway and can be used to find new therapeutics targeting Wnt/beta-catenin signaling.-Verkaar, F., Blankesteijn, W. M., Smits, J. F. M., Zaman, G. J. R. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin

  20. Nmp4/CIZ inhibits mechanically-induced β-catenin signaling activity in osteoblasts

    PubMed Central

    Yang, Zhouqi; Bidwell, Joseph P.; Young, Suzanne R.; Gerard-O’Riley, Rita; Wang, Haifang; Pavalko, Fredrick M.

    2010-01-01

    Cellular mechanotransduction, the process of converting mechanical signals into biochemical responses within cells, is a critical aspect of bone health. While the effects of mechanical loading on bone are well recognized, elucidating the specific molecular pathways involved in the processing of mechanical signals by bone cells represents a challenge and an opportunity to identify therapeutic strategies to combat bone loss. In this study we have for the first time examined the relationship between the nucleocytoplasmic shuttling transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ) and β-catenin signaling in response to a physiologic mechanical stimulation (oscillatory fluid shear stress, OFSS) in osteoblasts. Using calvaria-derived osteoblasts from Nmp4-deficient and wild-type mice, we found that the normal translocation of β-catenin to the nucleus in osteoblasts that is induced by OFSS is enhanced when Nmp4/CIZ is absent. Furthermore, we found that other aspects of OFSS-induced mechanotransduction generally associated with the β-catenin signaling pathway, including ERK, Akt and GSK3β activity, as well as expression of the β-catenin-responsive protein cyclin D1 are also enhanced in cells lacking Nmp4/CIZ. Finally, we found that in the absence of Nmp4/CIZ, OFSS-induced cytoskeletal reorganization and the formation of focal adhesions between osteoblasts and the extracellular substrate is qualitatively enhanced, suggesting that Nmp4/CIZ may reduce the sensitivity of bone cells to mechanical stimuli. Together these results provide experimental support for the concept that Nmp4/CIZ plays an inhibitory role in the response of bone cells to mechanical stimulation induced by OFSS. PMID:20112285

  1. [Differentiation of matrix proteins of influenza A viruses using enzyme immunoassay].

    PubMed

    Mohr, C; Döhner, L; Herrmann, B; Herrmann, H

    1978-01-01

    Matrix protein is known as a type-specific structural protein of influenza viruses. An attempt has been made to find out whether or not strain-specific components could be detected from matrix protein, in addition to its type-specific antigen determinants. The technique of enzyme immune assay was chosen as the optional method to differentiate between matrix proteins of various influenza-A viruses. Antigen titration was undertaken of several matrix proteins, using two specific anti-matrix-protein sera in each case. Information regarding serological relationships between the tested matrix proteins of various influenza-A viruses was obtained from a quotient between the titres of one antigen, on the one hand, and the two anti-matrix-protein sera used in titration, on the other. Two matrix protein sub-types were established in the context of the influenza-A viruses tested. Sub-type M1 was attributable to older strains (A/PR/8 and A/FM/1), whereas the matrix protein of sub-type M2 was found to be present in more recent strains (A/Hongkong and A/Port Chalmers).

  2. The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

    PubMed

    Miyamoto, Hiroshi; Endo, Hirotoshi; Hashimoto, Naoki; Limura, Kurin; Isowa, Yukinobu; Kinoshita, Shigeharu; Kotaki, Tomohiro; Masaoka, Tetsuji; Miki, Takumi; Nakayama, Seiji; Nogawa, Chihiro; Notazawa, Atsuto; Ohmori, Fumito; Sarashina, Isao; Suzuki, Michio; Takagi, Ryousuke; Takahashi, Jun; Takeuchi, Takeshi; Yokoo, Naoki; Satoh, Nori; Toyohara, Haruhiko; Miyashita, Tomoyuki; Wada, Hiroshi; Samata, Tetsuro; Endo, Kazuyoshi; Nagasawa, Hiromichi; Asakawa, Shuichi; Watabe, Shugo

    2013-10-01

    In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

  3. Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: reduction of Wnt-associated proteins and reduced translocation of galectin-3-mediated β-catenin to the nucleus.

    PubMed

    Dakeng, Sumana; Duangmano, Suwit; Jiratchariyakul, Weena; U-Pratya, Yaowalak; Bögler, Oliver; Patmasiriwat, Pimpicha

    2012-01-01

    The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and β-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules β-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3β levels. Cucurbitacin B treatment inhibited translocation to the nucleus of β-catenin and galectin-3. The depletion of β-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

  4. The WNT-less wonder: WNT-independent β-catenin signaling.

    PubMed

    Aktary, Zackie; Bertrand, Juliette U; Larue, Lionel

    2016-09-01

    β-catenin is known as an Armadillo protein that regulates gene expression following WNT pathway activation. However, WNT-independent pathways also activate β-catenin. During the establishment of the melanocyte lineage, β-catenin plays an important role. In the context of physiopathology, β-catenin is activated genetically or transiently in various cancers, including melanoma, where it can be found in the nucleus of tumors. In this review, we discuss alternative pathways that activate β-catenin independent of WNTs and highlight what is known regarding these pathways in melanoma. We also discuss the role of β-catenin as a transcriptional regulator in various cell types, with emphasis on the different transcription factors it associates with independent of WNT induction. Finally, the role of WNT-independent β-catenin in melanocyte development and melanomagenesis is also discussed. PMID:27311806

  5. Pea protein provides a promising matrix for microencapsulating iron.

    PubMed

    Bittencourt, Luciana Linhares de Azevedo; Pedrosa, Cristiana; Sousa, Valéria Pereira de; Pierucci, Anna Paola Trindade; Citelli, Marta

    2013-12-01

    Worldwide, the most prevalent nutritional deficiency is iron. The strategies for iron supplementation often fail due to poor adherence to supplementation methods contributed to unpleasant sensory characteristics. An alternative is the use of microencapsulated nutrients for home fortification in order to mask undesirable tastes and to allow its release in strategic sites of the gastrointestinal tract. Toward this end, pea protein concentrate was tested as a natural, edible and alternative material and the spray-drying technique was utilized for the preparation of microparticles containing ferrous sulfate. Their physical and chemical characteristics were evaluated. The microparticles had a spherical shape and grooves with an average size ranging between 2 and 3 μm. Analysis by in vitro assays tested the release of iron in simulated salivary and gastric fluids and its intestinal absorption in Caco-2 cells. No dissolution of iron occurred in the salivary medium whereas the sensory analysis showed good acceptance of a product which incorporated 5.5 mg of iron per 100 g portion of food. Thus, the effectiveness of microencapsulation was demonstrated by utilizing a plant protein as an encapsulating matrix for the controlled release of iron and capable of preserving the bioaccessibility of ferrous sulfate. PMID:23990387

  6. Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

    PubMed Central

    Bannai, Yuka; Aminova, Leila R.; Faulkner, Melinda J.; Ho, Mengfei; Wilson, Brenda A.

    2012-01-01

    The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling. PMID:22919671

  7. An Autoinhibited Structure of α-Catenin and Its Implications for Vinculin Recruitment to Adherens Junctions*

    PubMed Central

    Ishiyama, Noboru; Tanaka, Nobutoshi; Abe, Kentaro; Yang, Yoo Jeong; Abbas, Yazan M.; Umitsu, Masataka; Nagar, Bhushan; Bueler, Stephanie A.; Rubinstein, John L.; Takeichi, Masatoshi; Ikura, Mitsuhiko

    2013-01-01

    α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton. PMID:23589308

  8. Evidence for the Nucleo-Apical Shuttling of a Beta-Catenin Like Plasmodium falciparum Armadillo Repeat Containing Protein

    PubMed Central

    Mitra, Pallabi; Gupta, Enna Dogra; Sahar, Tajali; Pandey, Alok K.; Dangi, Poonam; Reddy, K. Sony; Chauhan, Virander Singh; Gaur, Deepak

    2016-01-01

    Eukaryotic Armadillo (ARM) repeat proteins are multifaceted with prominent roles in cell-cell adhesion, cytoskeletal regulation and intracellular signaling among many others. One such ARM repeat containing protein, ARM Repeats Only (ARO), has recently been demonstrated in both Toxoplasma (TgARO) and Plasmodium (PfARO) parasites to be targeted to the rhoptries during the late asexual stages. TgARO has been implicated to play an important role in rhoptry positioning i.e. directing the rhoptry towards the apical end of the parasite. Here, we report for the first time that PfARO exhibits a DNA binding property and a dynamic sub-cellular localization between the nucleus (early schizont) and rhoptry (late schizont) during the different stages of the asexual blood-stage life cycle. PfARO possesses a putative nuclear export signal (NES) and the nucleo-apical shuttling was sensitive to Leptomycin B (LMB) suggesting that the nuclear export was mediated by CRM1. Importantly, PfARO specifically bound an A-T rich DNA sequence of the P. falciparum Gyrase A (PfgyrA) gene, suggesting that the DNA binding specificity of PfARO is likely due to the AT-richness of the probe. This is a novel functional characteristic that has not been reported previously for any P. falciparum ARM containing protein and suggests a putative role for PfARO in gene regulation. This study describes for the first time a conserved P. falciparum ARM repeat protein with a high degree of functional versatility. PMID:26828945

  9. Evidence for the Nucleo-Apical Shuttling of a Beta-Catenin Like Plasmodium falciparum Armadillo Repeat Containing Protein.

    PubMed

    Mitra, Pallabi; Gupta, Enna Dogra; Sahar, Tajali; Pandey, Alok K; Dangi, Poonam; Reddy, K Sony; Chauhan, Virander Singh; Gaur, Deepak

    2016-01-01

    Eukaryotic Armadillo (ARM) repeat proteins are multifaceted with prominent roles in cell-cell adhesion, cytoskeletal regulation and intracellular signaling among many others. One such ARM repeat containing protein, ARM Repeats Only (ARO), has recently been demonstrated in both Toxoplasma (TgARO) and Plasmodium (PfARO) parasites to be targeted to the rhoptries during the late asexual stages. TgARO has been implicated to play an important role in rhoptry positioning i.e. directing the rhoptry towards the apical end of the parasite. Here, we report for the first time that PfARO exhibits a DNA binding property and a dynamic sub-cellular localization between the nucleus (early schizont) and rhoptry (late schizont) during the different stages of the asexual blood-stage life cycle. PfARO possesses a putative nuclear export signal (NES) and the nucleo-apical shuttling was sensitive to Leptomycin B (LMB) suggesting that the nuclear export was mediated by CRM1. Importantly, PfARO specifically bound an A-T rich DNA sequence of the P. falciparum Gyrase A (PfgyrA) gene, suggesting that the DNA binding specificity of PfARO is likely due to the AT-richness of the probe. This is a novel functional characteristic that has not been reported previously for any P. falciparum ARM containing protein and suggests a putative role for PfARO in gene regulation. This study describes for the first time a conserved P. falciparum ARM repeat protein with a high degree of functional versatility. PMID:26828945

  10. Activation of endothelial β-catenin signaling induces heart failure.

    PubMed

    Nakagawa, Akito; Naito, Atsuhiko T; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P; Adams, Ralf H; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  11. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  12. Disposable polymeric cryogel bioreactor matrix for therapeutic protein production.

    PubMed

    Jain, Era; Kumar, Ashok

    2013-05-01

    Low cost and high efficiency make disposable bioreactors feasible for small-scale therapeutic development and initial clinical trials. We have developed a cryogel-based disposable bioreactor matrix, which has been used for production of protein therapeutics such as urokinase and monoclonal antibodies (mAbs). The protocol discusses the application of a cryogel bioreactor for mAb production. Cryogels composed of either polyacrylamide (PAAm) coupled to gelatin or semi-interpenetrating PAAm-chitosan are synthesized by free-radical polymerization at -12 °C. Hybridoma cells are immobilized over the cryogel bioreactor and incubated for 48 h. Medium is circulated thereafter at 0.2 ml min(-1) and bioreactors can be run continuously for 60 d. The cryogel-based packed-bed bioreactor can be formulated as a monolith or as beads; it also has an efficiency four times what can be obtained using a tissue-culture flask, a high surface-to-volume ratio and effective nutrient transport. After incubation, the bioreactor setup will take about 60 min using a pre-prepared sterilized cryogel.

  13. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    PubMed

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development.

  14. Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions.

    PubMed

    Liljeroos, Lassi; Huiskonen, Juha T; Ora, Ari; Susi, Petri; Butcher, Sarah J

    2011-11-01

    Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix-matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.

  15. New Functions for Alpha-Catenins in Health and Disease: From Cancer to Heart Regeneration

    PubMed Central

    Vite, Alexia; Li, Jifen; Radice, Glenn L.

    2015-01-01

    Strong cell-cell adhesion mediated by adherens junctions is dependent on anchoring the transmembrane cadherin molecule to the underlying actin cytoskeleton. To do this, cadherin cytoplasmic domain interacts with catenin proteins, which include α-catenin that binds directly to filamentous actin. Originally thought to be a static structure, the connection between the cadherin/catenin adhesion complex and the actin cytoskeleton is now considered to be dynamic and responsive to both intercellular and intracellular signals. Alpha-catenins are mechanosensing proteins that undergo conformational change in response to cytoskeletal tension thus modifying the linkage between the cadherin and the actin cytoskeleton. There are three α-catenin isoforms expressed in mouse and human: αE-catenin (CTNNA1), αN-catenin (CTNNA2), and αT-catenin (CTNNA3). This review summarizes recent progress in understanding the in vivo function(s) of α-catenins in tissue morphogenesis, homeostasis, and disease. The role of α-catenin in the regulation of cellular proliferation will be discussed in the context of cancer and regeneration. PMID:25673211

  16. Pharmacological modulation of beta-catenin and its applications in cancer therapy

    PubMed Central

    Thakur, Ravi; Mishra, Durga Prasad

    2013-01-01

    Beta-catenin (β-catenin) is a multifunction protein with a central role in physiological homeostasis. Its abnormal expression leads to various diseases including cancer. In normal physiology, β-catenin either maintains integrity of epithelial tissues or controls transcription of various genes on extracellular instigations. In epithelial tissues, β-catenin functions as a component of the cadherin protein complex and regulates epithelial cell growth and intracellular adhesion. In Wnt signalling, β-catenin is a major transcriptional modulator and plays a crucial role in embryogenesis, stem cell renewal and organ regeneration. Aberrant expression of β-catenin can induce malignant pathways in normal cells and its abnormal activity is also exploited by existing malignant programmes. It acts as an oncogene and modulates transcription of genes to drive cancer initiation, progression, survival and relapse. Abnormal expression and function of β-catenin in cancer makes it a putative drug target. In the past decade, various attempts have been made to identify and characterize various pharmacological inhibitors of β-catenin. Many of these inhibitors are currently being investigated for their anticancer activities in a variety of cancers. The first half of this review will focus on the role of β-catenin in cancer initiation, maintenance, progression and relapse whereas the second half will briefly summarize the recent progress in development of agents for the pharmacological modulation of β-catenin activity in cancer therapeutics. PMID:23490077

  17. Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

    PubMed Central

    Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

    2012-01-01

    Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

  18. Diverse Basis of β-Catenin Activation in Human Hepatocellular Carcinoma: Implications in Biology and Prognosis

    PubMed Central

    Okabe, Hirohisa; Kinoshita, Hiroki; Imai, Katsunori; Nakagawa, Shigeki; Higashi, Takaaki; Arima, Kota; Uchiyama, Hideaki; Ikegami, Toru; Harimoto, Norifumi; Itoh, Shinji; Ishiko, Takatoshi; Yoshizumi, Tomoharu; Beppu, Toru; Monga, Satdarshan P. S.; Baba, Hideo; Maehara, Yoshihiko

    2016-01-01

    Aim β-catenin signaling is a major oncogenic pathway in hepatocellular carcinoma (HCC). Since β-catenin phosphorylation by glycogen synthase kinase 3β (GSK3β) and casein kinase 1ε (CK1ε) results in its degradation, mutations affecting these phosphorylation sites cause β-catenin stabilization. However, the relevance of missense mutations in non-phosphorylation sites in exon 3 remains unclear. The current study explores significance of such mutations in addition to addressing the clinical and biological implications of β-catenin activation in human HCC. Methods Gene alteration in exon3 of CTNNB1, gene expression of β-catenin targets such as glutamate synthetase (GS), axin2, lect2 and regucalcin (RGN), and protein expression of β-catenin were examined in 125 human HCC tissues. Results Sixteen patients (12.8%) showed conventional missense mutations affecting codons 33, 37, 41, and 45. Fifteen additional patients (12.0%) had other missense mutations in codon 32, 34, and 35. Induction of exon3 mutation caused described β-catenin target gene upregulation in HCC cell line. Interestingly, conventional and non-phosphorylation site mutations were equally associated with upregulation of β-catenin target genes. Nuclear localization of β-catenin was associated with poor overall survival (p = 0.0461). Of these patients with nuclear β-catenin localization, loss of described β-catenin target gene upregulation showed significant poorer overall survival than others (p = 0.0001). Conclusion This study suggests that both conventional and other missense mutations in exon 3 of CTNNB1 lead to β-catenin activation in human HCC. Additionally, the mechanism of nuclear β-catenin localization without upregulation of described β-catenin target genes might be of clinical importance depending on distinct mechanism. PMID:27100093

  19. Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

    PubMed Central

    Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

    2015-01-01

    The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

  20. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

    PubMed

    Münch, Christian; Harper, J Wade

    2016-06-30

    The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt. PMID:27350246

  1. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

    PubMed

    Münch, Christian; Harper, J Wade

    2016-06-30

    The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt.

  2. Insights into the Mechanisms Involved in the Expression and Regulation of Extracellular Matrix Proteins in Diabetic Nephropathy.

    PubMed

    Hu, C; Sun, L; Xiao, L; Han, Y; Fu, X; Xiong, X; Xu, X; Liu, Y; Yang, S; Liu, F; Kanwar, Y S

    2015-01-01

    Diabetic Nephropathy (DN) is believed to be a major microvascular complication of diabetes. The hallmark of DN includes deposition of Extracellular Matrix (ECM) proteins, such as, collagen, laminin and fibronectin in the mesangium and renal tubulo-interstitium of the glomerulus and basement membranes. Such an increased expression of ECM leads to glomerular and tubular basement membranes thickening and increase of mesangial matrix, ultimately resulting in glomerulosclerosis and tubulointerstitial fibrosis. The characteristic morphologic glomerular mesangial lesion has been described as Kimmelstiel-Wilson nodule, and the process at times is referred to as diabetic nodular glomerulosclerosis. Thus, the accumulation of ECM proteins plays a critical role in the development of DN. The relevant mechanism(s) involved in the increased ECM expression and their regulation in the kidney in diabetic state has been extensively investigated and documented in the literature. Nevertheless, there are certain other mechanisms that may yet be conclusively defined. Recent studies demonstrated that some of the new signaling pathways or molecules including, Notch, Wnt, mTOR, TLRs and small GTPase may play a pivotal role in the modulation of ECM regulation and expression in DN. Such modulation could be operational for instance Notch through Notch1/Jagged1 signaling, Wnt by Wnt/β- catenin pathway and mTOR via PI3-K/Akt/mTOR signaling pathways. All these pathways may be critical in the modulation of ECM expression and tubulo-interstitial fibrosis. In addition, TLRs, mainly the TLR2 and TLR4, by TLR2- dependent and TGF-β-dependent conduits, may modulate ECM expression and generate a fibrogenic response. Small GTPase like Rho, Ras and Rab family by targeting relevant genes may also influence the accumulation of ECM proteins and renal fibrosis in hyperglycemic states. This review summarizes the recent information about the role and mechanisms by which these molecules and signaling pathways

  3. Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions

    PubMed Central

    Liljeroos, Lassi; Huiskonen, Juha T.; Ora, Ari; Susi, Petri; Butcher, Sarah J.

    2011-01-01

    Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix–matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices. PMID:22025713

  4. Expression of e-cadherin, alpha-catenins and Beta-catenins in human gastric carcinomas - correlation with histology and tumor progression.

    PubMed

    Yasui, W; Kuniyasu, H; Akama, Y; Kitahara, K; Nagafuchi, A; Ishihara, S; Tsukita, S; Tahara, E

    1995-01-01

    The expression of cell-cell adhesion molecule, E-cadherin and its associated proteins, alpha- and beta-catenins in human gastric carcinomas was examined by Western blotting. All the seven gastric carcinoma cell lines expressed E-cadherin except KATOIII, which was derived from pleural effusion of a scirrhous type stomach cancer or Borrmann's type-4 carcinoma. The expression of alpha-catenin was not detected in HSC43 derived from scirrhous carcinoma, while HSC39 expressed abnormal beta-catenin caused by genetic alteration. In gastric carcinoma cases, the levels of E-cadherin and alpha-catenin were significantly lower in poorly differentiated adenocarcinomas and scirrhous carcinomas when compared to other types of gastric carcinomas. Deeply invasive carcinomas expressed E-cadherin and alpha-catenin at lower levels. However, the expression level of alpha-catenin was not necessarily consistent with that of E-cadherin. One of 10 gastric carcinomas examined showed complete deletion of alpha-catenin gene in Southern blotting. beta-catenin was expressed at lower level in poorly differentiated adenocarcinomas than in well-differentiated adenocarcinomas. These findings suggest that reduction in the expression of E-cadherin and its associated molecules, catenins, is involved in the development and infiltrative growth of scirrhous type gastric carcinomas. PMID:21597700

  5. Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth.

    PubMed

    Gong, Ningping; Shangguan, Junlong; Liu, Xiaojun; Yan, Zhenguang; Ma, Zhuojun; Xie, Liping; Zhang, Rongqing

    2008-10-01

    How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA-soluble matrix (SM) and EDTA-insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets.

  6. Syndecan-4 inhibits Wnt/β-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.

    PubMed

    Astudillo, Pablo; Carrasco, Héctor; Larraín, Juan

    2014-01-01

    Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/β-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/β-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/β-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling.

  7. Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies

    SciTech Connect

    van Wyke, K.L.; Yewdell, J.W.; Reck, L.J.; Murphy, B.R.

    1984-01-01

    Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. The authors found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of three epitopes are located in nonoverlapping domains on the matrix protein molecule.

  8. mRNA expression and protein localization of dentin matrix protein 1 during dental root formation.

    PubMed

    Toyosawa, S; Okabayashi, K; Komori, T; Ijuhin, N

    2004-01-01

    Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation. PMID:14751569

  9. Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth.

    PubMed

    Kim, Jung-Wook; Yamakoshi, Yasuo; Iwata, Takanori; Hu, Yuan Yuan; Zhang, Hengmin; Hu, Jan C-C; Simmer, James P

    2006-02-01

    Dentin matrix protein 1 (DMP1) is an acidic non-collagenous protein that is necessary for the proper biomineralization of bone, cartilage, cementum, dentin, and enamel. Dentin matrix protein 1 is highly phosphorylated and potentially glycosylated, but there is no experimental data identifying which specific amino acids are modified. For the purpose of facilitating the characterization of DMP1 from pig, which has the advantage of large developing teeth for obtaining protein in quantity and extensive structural information concerning other tooth matrix proteins, we characterized the porcine DMP1 cDNA and gene structure, raised anti-peptide immunoglobulins that are specific for porcine DMP1, and detected DMP1 protein in porcine tooth extracts and histological sections. Porcine DMP1 has 510 amino acids, including a 16-amino acid signal peptide. The deduced molecular weight of the secreted, unmodified protein is 53.5 kDa. The protein has 93 serines and 12 threonines in the appropriate context for phosphorylation, and four asparagines in a context suitable for glycosylation. Dentin matrix protein 1 protein bands with apparent molecular weights between 30 and 45 kDa were observed in partially purified dentin extracts. In developing teeth, immunohistochemistry localized DMP1 in odontoblasts and the dentinal tubules of mineralized dentin and in ameloblasts, but not in the enamel matrix.

  10. Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

    PubMed Central

    Montes de Oca, Addy; Guerrero, Fatima; Martinez-Moreno, Julio M.; Madueño, Juan A.; Herencia, Carmen; Peralta, Alan; Almaden, Yolanda; Lopez, Ignacio; Aguilera-Tejero, Escolastico; Gundlach, Kristina; Büchel, Janine; Peter, Mirjam E.; Passlick-Deetjen, Jutta; Rodriguez, Mariano; Muñoz-Castañeda, Juan R.

    2014-01-01

    Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro

  11. Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation

    PubMed Central

    Coluccia, Addolorata Maria Luce; Vacca, Angelo; Duñach, Mireia; Mologni, Luca; Redaelli, Sara; Bustos, Victor H; Benati, Daniela; Pinna, Lorenzo A; Gambacorti-Passerini, Carlo

    2007-01-01

    Self-renewal of Bcr-Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated β-catenin. Although β-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of β-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with β-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β-catenin at Y86 and Y654 residues. This Y-phospho β-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the β-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated β-catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr-Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β-catenin Y phosphorylation, Axin/GSK3β binding to β-catenin and its subsequent S/T phosphorylation. Silencing of β-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of β-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML. PMID:17318191

  12. FANCL ubiquitinates β-catenin and enhances its nuclear function

    PubMed Central

    Rotelli, Michael D.; Petersen, Curtis L.; Kaech, Stefanie; Nelson, Whitney D.; Yates, Jane E.; Hanlon Newell, Amy E.; Olson, Susan B.; Druker, Brian J.; Bagby, Grover C.

    2012-01-01

    Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34+ stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss. PMID:22653977

  13. Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma.

    PubMed

    Annavarapu, Srinivas R; Cialfi, Samantha; Dominici, Carlo; Kokai, George K; Uccini, Stefania; Ceccarelli, Simona; McDowell, Heather P; Helliwell, Timothy R

    2013-10-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

  14. Characterization of Wnt/β-catenin signaling in rhabdomyosarcoma.

    PubMed

    Annavarapu, Srinivas R; Cialfi, Samantha; Dominici, Carlo; Kokai, George K; Uccini, Stefania; Ceccarelli, Simona; McDowell, Heather P; Helliwell, Timothy R

    2013-10-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. β-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/β-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed β-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of β-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of β-catenin, stabilization of the active cytosolic form and nuclear translocation of β-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/β-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target. PMID:23999248

  15. PEX5, the shuttling import receptor for peroxisomal matrix proteins, is a redox-sensitive protein.

    PubMed

    Apanasets, Oksana; Grou, Cláudia P; Van Veldhoven, Paul P; Brees, Chantal; Wang, Bo; Nordgren, Marcus; Dodt, Gabriele; Azevedo, Jorge E; Fransen, Marc

    2014-01-01

    Peroxisome maintenance depends on the import of nuclear-encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C-terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N-terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox-sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination-deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed. PMID:24118911

  16. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  17. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures.

    PubMed

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A; Harris, William A

    2013-04-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

  18. Interplay of matrix stiffness and protein tethering in stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Wen, Jessica H.; Vincent, Ludovic G.; Fuhrmann, Alexander; Choi, Yu Suk; Hribar, Kolin C.; Taylor-Weiner, Hermes; Chen, Shaochen; Engler, Adam J.

    2014-10-01

    Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

  19. Interplay of matrix stiffness and protein tethering in stem cell differentiation.

    PubMed

    Wen, Jessica H; Vincent, Ludovic G; Fuhrmann, Alexander; Choi, Yu Suk; Hribar, Kolin C; Taylor-Weiner, Hermes; Chen, Shaochen; Engler, Adam J

    2014-10-01

    Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

  20. Matrix Gla Protein polymorphism, but not concentrations, is associated with radiographic hand osteoarthritis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective. Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evalua...

  1. Ultrastructural localization of extracellular matrix proteins in liver biopsies using ultracryomicrotomy and immuno-gold labelling.

    PubMed

    Burt, A D; Griffiths, M R; Schuppan, D; Voss, B; MacSween, R N

    1990-01-01

    We describe a technique for the localization of extracellular matrix proteins in wedge and needle biopsy specimens of human liver. Using ultra-thin (50-70 nm) sections of puncture perfusion fixed tissue, extracellular matrix proteins were localized using a protein A-gold labelling procedure. We obtained good preservation of ultrastructural detail and specific labelling for collagen types I, III and IV and fibronectin. The method represents a sensitive means of identifying the extracellular distribution and the cellular origin of these proteins in normal and diseased human liver.

  2. EFEMP1 is repressed by estrogen and inhibits the epithelial-mesenchymal transition via Wnt/β-catenin signaling in endometrial carcinoma

    PubMed Central

    Qiu, Haifeng; Li, Bilan; Wang, Jingyun; Du, Guiqiang; Ren, Chune; Wan, Xiaoping

    2016-01-01

    Epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) acted as a tumor suppressor in endometrial carcinoma (EC). However, the correlation between EFEMP1 and estrogen is unknown. Here, we reported that the expression of EFEMP1 was conversely associated with ERα in endometrial carcinoma tissues. In endometrial carcinoma cells, estrogen/ERα signaling significantly suppressed the expression of EFEMP1. Moreover, chromatin immunoprecipitation (CHIP) and dual-luciferase reporter assays demonstrate that estrogen/ERα bound to the estrogen response element (ERE) located in EFEMP1 promoter and repressed its expression. Besides, in vitro and in vivo, EFEMP1 could remarkably suppress the expression of epithelial-mesenchymal transition (EMT) markers such as Vimentin, Snail and the Wnt/β-catenin target genes like Cyclin-D1 and c-Myc, which could be restored when EFEMP1 was silenced. In addition, XAV93920 (the inhibitor of the Wnt/β-catenin pathway) blocked and LiCl (the activator of the Wnt/β-catenin pathway) enhanced the effect of EFEMP1 on EMT. In conclusion, we demonstrated that estrogen/ERα signal suppresses EFEMP1. Besides, EFEMP1 inhibits EMT via interfering the Wnt/β-catenin signaling. PMID:27015552

  3. FRZB knockdown upregulates β-catenin activity and enhances cell aggressiveness in gastric cancer.

    PubMed

    Qin, Shuai; Zhang, Zhuo; Li, Jianfang; Zang, Lu

    2014-05-01

    Studies have shown that FRZB correlates with gastric tumorigenicity and may play role in regulating the Wnt/β‑catenin signaling pathway. In the present study, we investigated the correlation between FRZB and the Wnt/β‑catenin signaling pathway using gastric cancer tissues and an FRZB‑knockdown gastric cancer cell line model. The protein levels of FRZB and β‑catenin were examined using immunohistochemical staining. FRZB-specific shRNAs were used to generate FRZB‑knockdown MKN45 gastric cancer cells. Cell proliferation assay, suspending culture and Annexin V/PI double staining analysis were used to investigate the role of FRZB knockdown in cell growth. In vitro migration/invasion assays were performed. The expression of Wnt/β‑catenin downstream targets was analyzed by RT-PCR. FRZB mRNA levels showed negative correlation with β‑catenin levels in paired non-tumor and tumor tissues. FRZB protein levels were negatively correlated with β‑catenin levels analyzed by IHC staining. Furthermore, high FRZB protein levels were correlated with membrane localization of β‑catenin. FRZB knockdown increased gastric cancer cell growth in monolayer and soft agar culture; it increased cell aggregates in suspending culture and rendered less apoptosis which indicated increased anti-anoikis growth. FRZB knockdown increased cell migration and invasion and increased the expression of Wnt/β‑catenin downstream targets such as MMP7 and cyclin D1. Our studies revealed that FRZB levels were correlated with β‑catenin subcellular localization. Knockdown of FRZB in gastric cancer cells increased cell growth and migration/invasion which was also accompanied by activation of Wnt/β‑catenin downstream targets. FRZB knockdown may upregulate the Wnt/β‑catenin pathway and promote aggressiveness in gastric cancer. PMID:24676361

  4. [Electron transfer between globular proteins. Evaluation of a matrix element].

    PubMed

    Lakhno, V D; Chuev, G N; Ustinin, M N

    1998-01-01

    The dependence of the matrix element of the probability of interprotein electron transfer on the mutual orientation of the donor and acceptor centers and the distance between them was calculated. The calculations were made under the assumption that electron transfer proceeds mainly by a collective excitation of polaron nature, like a solvated electron state. The results obtained are consistent with experimental data and indicate the nonexponential behavior of this dependence in the case when the distance transfer is less than 20 A.

  5. Kinetics of protein-protein complex coacervation and biphasic release of salbutamol sulfate from coacervate matrix.

    PubMed

    Tiwari, Ananya; Bindal, Sonal; Bohidar, H B

    2009-01-12

    Turbidimetric titration was used to initiate associative intermolecular interactions between a pair of protein molecules, gelatin-A and gelatin-B, having complementary charges that led to pH-induced liquid-liquid phase separation and the formation of complex coacervate. The stoichiometric binding ratio was found to be [gelatin-A]/[gelatin-B]=3:2. The size of soluble intermolecular aggregates present in the supernatant exhibited interesting time-dependent coacervation because of residual electrostatic interactions. Dynamic light scattering and turbidity studies provided a systematic account of coacervation behavior. Rheology studies attributed the softening of the coacervate matrix to the presence of encapsulated salbutamol sulfate. The in vitro drug release kinetics was probed in simulated gastric fluid medium at physiological temperature (37 degrees C), which showed biphasic behavior. The initial release kinetics exhibited an exponential growth to saturation behavior, followed by a slower logarithmic release process.

  6. Non-collagenous protein screening in the human chondrodysplasias: link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin.

    PubMed

    Stanescu, V; Do, T P; Chaminade, F; Maroteaux, P; Stanescu, R

    1994-05-15

    A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudoachondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia. PMID:8030664

  7. Detecting protein-protein interactions with a novel matrix-based protein sequence representation and support vector machines.

    PubMed

    You, Zhu-Hong; Li, Jianqiang; Gao, Xin; He, Zhou; Zhu, Lin; Lei, Ying-Ke; Ji, Zhiwei

    2015-01-01

    Proteins and their interactions lie at the heart of most underlying biological processes. Consequently, correct detection of protein-protein interactions (PPIs) is of fundamental importance to understand the molecular mechanisms in biological systems. Although the convenience brought by high-throughput experiment in technological advances makes it possible to detect a large amount of PPIs, the data generated through these methods is unreliable and may not be completely inclusive of all possible PPIs. Targeting at this problem, this study develops a novel computational approach to effectively detect the protein interactions. This approach is proposed based on a novel matrix-based representation of protein sequence combined with the algorithm of support vector machine (SVM), which fully considers the sequence order and dipeptide information of the protein primary sequence. When performed on yeast PPIs datasets, the proposed method can reach 90.06% prediction accuracy with 94.37% specificity at the sensitivity of 85.74%, indicating that this predictor is a useful tool to predict PPIs. Achieved results also demonstrate that our approach can be a helpful supplement for the interactions that have been detected experimentally. PMID:26000305

  8. 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.

  9. MEKK2 mediates an alternative β-catenin pathway that promotes bone formation

    PubMed Central

    Greenblatt, Matthew Blake; Shin, Dong Yeon; Oh, Hwanhee; Lee, Ki-Young; Zhai, Bo; Gygi, Steven P.; Lotinun, Sutada; Baron, Roland; Liu, Dou; Su, Bing; Glimcher, Laurie H.; Shim, Jae-Hyuck

    2016-01-01

    Proper tuning of β-catenin activity in osteoblasts is required for bone homeostasis, because both increased and decreased β-catenin activity have pathologic consequences. In the classical pathway for β-catenin activation, stimulation with WNT ligands suppresses constitutive phosphorylation of β-catenin by glycogen synthase kinase 3β, preventing β-catenin ubiquitination and proteasomal degradation. Here, we have found that mitogen-activated protein kinase kinase kinase 2 (MAP3K2 or MEKK2) mediates an alternative pathway for β-catenin activation in osteoblasts that is distinct from the canonical WNT pathway. FGF2 activates MEKK2 to phosphorylate β-catenin at serine 675, promoting recruitment of the deubiquitinating enzyme, ubiquitin-specific peptidase 15 (USP15). USP15 in turn prevents the basal turnover of β-catenin by inhibiting its ubiquitin-dependent proteasomal degradation, thereby enhancing WNT signaling. Analysis of MEKK2-deficient mice and genetic interaction studies between Mekk2- and β-catenin–null alleles confirm that this pathway is an important physiologic regulator of bone mass in vivo. Thus, an FGF2/MEKK2 pathway mediates an alternative nonclassical pathway for β-catenin activation, and this pathway is a key regulator of bone formation by osteoblasts. PMID:26884171

  10. Characterization of the Unconventional Secretion of the Ebola Matrix Protein VP40.

    PubMed

    Reynard, Olivier; Mateo, Mathieu

    2016-01-01

    While most secreted proteins use the classical endoplasmic reticulum (ER)-Golgi secretion pathway to reach the extracellular medium, a few proteins are secreted through unconventional secretary pathways. Viral proteins can be secreted through unconventional secretion pathways. Here, we describe how we have recently demonstrated that the Ebola virus (EBOV) matrix protein VP40 is released from transfected and infected cells in a soluble form through an unconventional secretion pathway. PMID:27665561

  11. Zona localization of shell matrix proteins in mantle of Haliotis tuberculata (Mollusca, Gastropoda).

    PubMed

    Jolly, Cécile; Berland, Sophie; Milet, Christian; Borzeix, Sandrine; Lopez, Evelyne; Doumenc, Dominique

    2004-01-01

    Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.

  12. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc.

    PubMed

    Bedore, Jake; Leask, Andrew; Séguin, Cheryle A

    2014-07-01

    The so-called "matricellular" proteins have recently emerged as important regulators of cell-extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell-ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.

  13. Cadherins and catenins in dendrite and synapse morphogenesis

    PubMed Central

    Seong, Eunju; Yuan, Li; Arikkath, Jyothi

    2015-01-01

    Neurons are highly polarized specialized cells. Neuronal integrity and functional roles are critically dependent on dendritic architecture and synaptic structure, function and plasticity. The cadherins are glycosylated transmembrane proteins that form cell adhesion complexes in various tissues. They are associated with a group of cytosolic proteins, the catenins. While the functional roles of the complex have been extensively investigates in non-neuronal cells, it is becoming increasingly clear that components of the complex have critical roles in regulating dendritic and synaptic architecture, function and plasticity in neurons. Consistent with these functional roles, aberrations in components of the complex have been implicated in a variety of neurodevelopmental disorders. In this review, we discuss the roles of the classical cadherins and catenins in various aspects of dendrite and synapse architecture and function and their relevance to human neurological disorders. Cadherins are glycosylated transmembrane proteins that were initially identified as Ca2+-dependent cell adhesion molecules. They are present on plasma membrane of a variety of cell types from primitive metazoans to humans. In the past several years, it has become clear that in addition to providing mechanical adhesion between cells, cadherins play integral roles in tissue morphogenesis and homeostasis. The cadherin family is composed of more than 100 members and classified into several subfamilies, including classical cadherins and protocadherins. Several of these cadherin family members have been implicated in various aspects of neuronal development and function.1-3 The classical cadherins are associated with a group of cytosolic proteins, collectively called the catenins. While the functional roles of the cadherin-catenin cell adhesion complex have been extensively investigated in epithelial cells, it is now clear that components of the complex are well expressed in central neurons at different

  14. Endoplasmic Reticulum Chaperone Protein GRP-78 Mediates Endocytosis of Dentin Matrix Protein 1*S⃞

    PubMed Central

    Ravindran, Sriram; Narayanan, Karthikeyan; Eapen, Asha Sarah; Hao, Jianjun; Ramachandran, Amsaveni; Blond, Sylvie; George, Anne

    2008-01-01

    Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant KD = 85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development. PMID:18757373

  15. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  16. Towards a matrix mechanics framework for dynamic protein network

    PubMed Central

    2010-01-01

    Protein–protein interaction networks are currently visualized by software generated interaction webs based upon static experimental data. Current state is limited to static, mostly non-compartmental network and non time resolved protein interactions. A satisfactory mathematical foundation for particle interactions within a viscous liquid state (situation within the cytoplasm) does not exist nor do current computer programs enable building dynamic interaction networks for time resolved interactions. Building mathematical foundation for intracellular protein interactions can be achieved in two increments (a) trigger and capture the dynamic molecular changes for a select subset of proteins using several model systems and high throughput time resolved proteomics and, (b) use this information to build the mathematical foundation and computational algorithm for a compartmentalized and dynamic protein interaction network. Such a foundation is expected to provide benefit in at least two spheres: (a) understanding physiology enabling explanation of phenomenon such as incomplete penetrance in genetic disorders and (b) enabling several fold increase in biopharmaceutical production using impure starting materials. PMID:20805933

  17. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    PubMed Central

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone. PMID:22573329

  18. Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1

    PubMed Central

    Knoops, Kèvin; de Boer, Rinse; Kram, Anita

    2015-01-01

    Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high-resolution electron microscopy techniques. We observed that the cells contained peroxisomal membrane remnants, which in ultrathin cross sections generally appeared as double membrane rings. Electron tomography revealed that these structures consisted of one continuous membrane, representing an empty, flattened vesicle, which folds into a cup shape. Immunocytochemistry revealed that these structures lack peroxisomal matrix proteins but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13, and Pex14. Upon reintroduction of Pex1 in Pex1-deficient cells, these peroxisomal membrane remnants (ghosts) rapidly incorporated peroxisomal matrix proteins and developed into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import. PMID:26644511

  19. β-Catenin Signaling and Roles in Liver Homeostasis, Injury, and Tumorigenesis

    PubMed Central

    Monga, Satdarshan Pal

    2015-01-01

    β -catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway; alterations in its activity have been associated with the development of hepatocellular carcinoma and other liver diseases. Other than WNT, additional signaling pathways also can converge at β-catenin. β-catenin also interacts with transcription factors such as T-cell factor, forkhead box protein O, and hypoxia inducible factor 1α to regulate the expression of target genes. We discuss the role of β-catenin in metabolic zonation of the adult liver. β-catenin also regulates the expression of genes that control metabolism of glucose, nutrients, and xenobiotics; alterations in its activity may contribute to the pathogenesis of nonalcoholic steatohepatitis. Alterations in β-catenin signaling may lead to activation of hepatic stellate cells, which is required for fibrosis. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in CTNNB1 that result in constitutive activation of β-catenin, so this molecule could be a therapeutic target. We discuss how alterations in β-catenin activity contribute to liver disease and how these might be used in diagnosis and prognosis, as well as in the development of therapeutics. PMID:25747274

  20. Expression and prognostic value of SFRP1 and β-catenin in patients with glioblastoma

    PubMed Central

    CHANG, LIANG; LEI, XUHUI; QIN, YU; ZENG, GUANGCHUN; ZHANG, XUEXIN; JIN, HUA; WANG, CHAO; WANG, XIN; SU, JUN

    2016-01-01

    The roles of secreted frizzled-related protein-1 (SFRP1) and β-catenin in human cancer have been widely studied, and it has recently been demonstrated that these proteins are associated with numerous human carcinomas. However, their clinical significance in glioblastoma multiforme (GBM) has not been examined. The current study aimed to analyze the correlation between the expression of SFRP1 and β-catenin, and clinicopathological characteristics in GBM patients. The expression of SFRP1 and β-catenin was assessed by immunohistochemistry in 113 samples of GBM and 40 normal brain tissues. Compared with normal brain tissues, GBM tissues exhibited significantly lower expression of SFRP1, and higher expression of β-catenin (both P<0.05). A Kaplan-Meier analysis revealed that patients with positive SFRP1 expression had a significantly longer overall survival (OS) time relative to those with negative SFRP1 expression (P<0.000), and that patients with positive β-catenin expression had a shorter OS time than those with negative β-catenin expression (P<0.000). A multivariate Cox regression analysis indicated that adjuvant treatment, SFRP1 expression and β-catenin expression were independent prognostic factors for OS (P<0.000, P=0.008 and P=0.001, respectively) in patients with GBM. The current data suggest that expression of SFRP1 and β-catenin may be considered significant prognostic indicators for patients with GBM. PMID:26870169

  1. Rhabdovirus matrix protein structures reveal a novel mode of self-association.

    PubMed

    Graham, Stephen C; Assenberg, René; Delmas, Olivier; Verma, Anil; Gholami, Alireza; Talbi, Chiraz; Owens, Raymond J; Stuart, David I; Grimes, Jonathan M; Bourhy, Hervé

    2008-12-01

    The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

  2. Beta-catenin in disease

    PubMed Central

    Prakash, Sharada; Swaminathan, Uma; Nagamalini, BR; Krishnamurthy, Ashwini Balkuntla

    2016-01-01

    In continuation with the previous review on “β-catenin in health”, in this review we discuss the role of β-catenin in the pathogenesis of common oral lesions in the oral and maxillofacial region- oral potentially malignant disorders, their progression to oral squamous cell carcinoma, salivary gland tumors and odontogenic tumours. This review is based on a pubmed search of all the lesions included in the review. PMID:27601825

  3. Beta-catenin in disease

    PubMed Central

    Prakash, Sharada; Swaminathan, Uma; Nagamalini, BR; Krishnamurthy, Ashwini Balkuntla

    2016-01-01

    In continuation with the previous review on “β-catenin in health”, in this review we discuss the role of β-catenin in the pathogenesis of common oral lesions in the oral and maxillofacial region- oral potentially malignant disorders, their progression to oral squamous cell carcinoma, salivary gland tumors and odontogenic tumours. This review is based on a pubmed search of all the lesions included in the review.

  4. Beta-catenin in disease.

    PubMed

    Prakash, Sharada; Swaminathan, Uma; Nagamalini, B R; Krishnamurthy, Ashwini Balkuntla

    2016-01-01

    In continuation with the previous review on "β-catenin in health", in this review we discuss the role of β-catenin in the pathogenesis of common oral lesions in the oral and maxillofacial region- oral potentially malignant disorders, their progression to oral squamous cell carcinoma, salivary gland tumors and odontogenic tumours. This review is based on a pubmed search of all the lesions included in the review. PMID:27601825

  5. Intrinsic fluorescence excitation-emission matrix spectral features of cottonseed protein fractions and the effects of denaturants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To better understand the functional and physicochemical properties of cottonseed protein, we investigated the intrinsic fluorescence excitation-emission matrix (EEM) spectral features of cottonseed protein isolate (CSPI) and sequentially extracted water (CSPw) and alkali (CSPa) protein fractions, an...

  6. Articular-cartilage matrix gamma-carboxyglutamic acid-containing protein. Characterization and immunolocalization.

    PubMed Central

    Loeser, R; Carlson, C S; Tulli, H; Jerome, W G; Miller, L; Wallin, R

    1992-01-01

    Matrix gamma-carboxyglutamic acid (Gla)-containing protein (MGP) was found to be present in articular cartilage by Western-blot analysis of guanidinium chloride extracts of human and bovine cartilage and was further localized by immunohistochemical studies on human and monkey specimens. In newborn articular cartilage MGP was present diffusely throughout the matrix, whereas in growth-plate cartilage it was seen mainly in late hypertrophic and calcifying-zone chondrocytes. In adult articular cartilage MGP was present primarily in chondrocytes and the pericellular matrix. Immunoelectron microscopy studies revealed an association between MGP and vesicular structures with an appearance consistent with matrix vesicles. MGP may be an important regulator of cartilage calcification because of its localization in cartilage and the known affinity of Gla-containing proteins for Ca2+ and hydroxyapatite. Images Fig. 1. Fig. 2. Fig. 3. PMID:1540125

  7. Rabies virus matrix protein induces apoptosis by targeting mitochondria.

    PubMed

    Zan, Jie; Liu, Juan; Zhou, Jian-Wei; Wang, Hai-Long; Mo, Kai-Kun; Yan, Yan; Xu, Yun-Bin; Liao, Min; Su, Shuo; Hu, Rong-Liang; Zhou, Ji-Yong

    2016-09-10

    Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination. PMID:27426727

  8. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

    PubMed

    Sun, Yan; Jallerat, Quentin; Szymanski, John M; Feinberg, Adam W

    2015-02-01

    Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

  9. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    PubMed

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  10. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins.

    PubMed

    Capossela, S; Schläfli, P; Bertolo, A; Janner, T; Stadler, B M; Pötzel, T; Baur, M; Stoyanov, J V

    2014-04-04

    Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

  11. The Wnt/beta-catenin pathway is activated during advanced arterial aging in humans.

    PubMed

    Marchand, Alexandre; Atassi, Fabrice; Gaaya, Amira; Leprince, Pascal; Le Feuvre, Claude; Soubrier, Florent; Lompré, Anne-Marie; Nadaud, Sophie

    2011-04-01

    Aging is the main risk factor for cardiovascular diseases, but the associated molecular mechanisms are poorly understood. The Wnt signaling pathway was shown to be induced during aging in muscle and in the skin, but the regulation and role of Wnt signaling in the aged vessel have not yet been addressed. While screening for age-related changes in gene expression in the intima/media of human mammary arteries, we observed that the expression of frizzled 4 (Fzd4), a Wnt receptor, and of several targets of the Wnt/β-catenin/TCF signaling pathway [Wnt-inducible secreted protein 1 (WISP1), versican, osteopontin (SPP1), insulin-like growth factor binding protein 2 (IGFBP-2), and p21] were modified with age, suggesting an activation of the Wnt/β-catenin pathway. In contrast, we did not observe any regulation of forkhead transcription factor (FoxO) target genes. Beta-catenin-activating phosphorylation at position Ser675 was increased in aging mammary arteries, confirming the activation of this pathway. We confirmed in vitro that Wnt3a or Wnt1 treatment of human vascular smooth muscle cells (VSMCs) induced β-catenin phosphorylation at Ser675 and WISP1, SPP1, and IGFBP-2 expression. In vitro, Wnt treatment induced proliferation and cyclin D1 expression in VSMC from young (6 weeks old) rats but not in cells from older rats (8 months old), even though low-density lipoprotein receptor-related protein 6 and β-catenin phosphorylation, and β-catenin nuclear translocation demonstrated β-catenin activation in both cell types. Beta-catenin silencing demonstrated that Wnt induction of cyclin D1 expression is β-catenin dependent. Altogether, our data show that the Wnt/β-catenin/TCF pathway is activated in aging human mammary artery cells, but fails to induce the proliferation of aging vascular cells. PMID:21108734

  12. Activation of the wnt/β-Catenin Signaling Pathway in Polymyositis, Dermatomyositis and Duchenne Muscular Dystrophy

    PubMed Central

    Liu, Fuchen; Liang, Zonglai; Xu, Jingwen; Li, Wei; Zhao, Dandan; Zhao, Yuying

    2016-01-01

    Background and Purpose The wnt/β-catenin signaling pathway plays a critical role in embryonic development and adult-tissue homeostasis. Recent investigations implicate the importance of wnt/β-catenin signaling in normal wound healing and its sustained activation being associated with fibrogenesis. We investigated the immunolocalization and activation of wnt/β-catenin in polymyositis (PM), dermatomyositis (DM), and Duchenne muscular dystrophy (DMD). Methods Immunofluorescence staining and Western blot analysis of β-catenin were performed in muscle specimens from 6 PM, 8 DM, and 6 DMD subjects. The β-catenin/Tcf4 DNA-binding activity in muscle was studied using an electrophoretic mobility shift assay (EMSA), and serum wnt/β-catenin/Tcf transcriptional activity was measured using a luciferase reporter gene assay. Results Immunoreactivity for β-catenin was found in the cytoplasm and nuclei of muscle fibers in PM, DM, and DMD. The protein level of β-catenin was elevated, and EMSA analysis confirmed the activation of wnt/β-catenin signaling. The transcriptional activities of β-catenin/Tcf in the circulation were increased in patients with PM, DM, and DMD, especially in those with interstitial lung disease, and these transcriptional activities decreased when PM or DM patients exhibited obvious clinical improvements. Conclusions Our findings indicate that wnt/β-catenin signaling is activated in PM, DM, and DMD. Its activation in muscle tissue and the circulation may play a role in modulating muscle regeneration and be at least partly involved in the process of muscle and pulmonary fibrosis. PMID:27165423

  13. Expression and colocalization of β-catenin and lymphoid enhancing factor-1 in prostate cancer progression.

    PubMed

    Bauman, Tyler M; Vezina, Chad M; Ricke, Emily A; Halberg, Richard B; Huang, Wei; Peterson, Richard E; Ricke, William A

    2016-05-01

    The purpose of this study was to objectively investigate β-catenin and LEF1 abundance, subcellular localization, and colocalization across benign and staged prostate cancer (PCa) specimens. A tissue microarray containing tumor-adjacent histologically benign prostate tissue (BPT; n = 48 patients), high-grade prostatic intraepithelial neoplasia (HGPIN; n = 25), localized PCa (n = 42), aggressive PCa (n = 31), and metastases (n = 22) was stained using multiplexed immunohistochemistry with antibodies toward E-cadherin, β-catenin, and LEF1. Multispectral imaging was used for quantitation, and protein expression and colocalization was evaluated across PCa progression. Stromal nuclear β-catenin abundance was greater in HGPIN and PCa compared with BPT (P < .05 for both), and epithelial nuclear β-catenin abundance was lower in metastatic PCa than in BPT (P < .05 for both). Epithelial and stromal nuclear LEF1 abundance was greater in HGPIN compared with BPT, whereas epithelial nuclear LEF1 was also greater in metastases. The proportion of epithelial and stromal nuclear double-positive β-catenin(+)/LEF1(+) cells was greater in HGPIN compared with BPT. In addition, the proportion of epithelial β-catenin(+)/LEF1(+) cells was greater in localized PCa and metastases compared with BPT. A significant amount of stromal cells were positive for LEF1 but not β-catenin. β-Catenin and LEF1 abundance were negatively correlated in the epithelium (P < .0001) but not the stroma (P > .05). We conclude that β-catenin and LEF1 colocalization is increased in HGPIN and metastasis relative to BPT, suggesting a role for β-catenin/LEF1-mediated transcription in both malignant transformation and metastasis of PCa. Furthermore, our results suggest that LEF1 abundance alone is not a reliable readout for β-catenin activity in prostate tissues.

  14. Novel proteins identified in the insoluble byssal matrix of the freshwater zebra mussel.

    PubMed

    Gantayet, Arpita; Rees, David J; Sone, Eli D

    2014-04-01

    The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion. PMID:24057171

  15. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  16. Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/β-catenin signaling pathway

    PubMed Central

    Mercer, Kelly E.; Hennings, Leah; Sharma, Neha; Lai, Keith; Cleves, Mario A.; Wynne, Rebecca A.; Badger, Thomas M.; Ronis, Martin J.J.

    2014-01-01

    Although alcohol effects within the liver have been extensively studied, the complex mechanisms by which alcohol causes liver cancer are not well understood. It has been suggested that ethanol (EtOH) metabolism promotes tumor growth by increasing hepatocyte proliferation. In this study, we developed a mouse model of tumor promotion by chronic EtOH consumption in which EtOH feeding began 46 days post-injection of the chemical carcinogen diethylnitrosamine (DEN) and continued for 16 weeks. With a final EtOH concentration of 28% of total calories, we observed a significant increase in the total number of preneoplastic foci and liver tumors per mouse in the EtOH+DEN group compared to corresponding pair-fed (PF)+DEN and chow+DEN control groups. We also observed a 4-fold increase in hepatocyte proliferation (p<0.05) and increased cytoplasmic staining of active-β-catenin in non-tumor liver sections from EtOH+DEN mice compared to PF+DEN controls. In a rat model of alcohol-induced liver disease, we found increased hepatocyte proliferation (p<0.05); depletion of retinol and retinoic acid stores (p<0.05); increased expression of cytosolic and nuclear expression of β-catenin (p<0.05) and phosphorylated-glycogen synthase kinase 3 β (p-GSK3β, p<0.05; significant up-regulation in Wnt7a mRNA expression; and increased expression of several β-catenin targets, including, glutamine synthetase (GS), cyclin D1, Wnt1 inducible signaling pathway protein (WISP1), and matrix metalloproteinase-7 (MMP7), p<0.05. These data suggest that chronic EtOH consumption activates the Wnt/β-catenin signaling pathways to increase hepatocyte proliferation, thus promoting tumorigenesis following an initiating insult to the liver. PMID:24778325

  17. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  18. Extracellular matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries.

    PubMed Central

    Botney, M. D.; Kaiser, L. R.; Cooper, J. D.; Mecham, R. P.; Parghi, D.; Roby, J.; Parks, W. C.

    1992-01-01

    Lobar pulmonary arteries from patients with unexplained pulmonary hypertension were obtained at the time of single-lung transplantation to determine the response of large elastic vessels to increased intraluminal pressure. Specifically, human pulmonary arteries were examined to determine if remodeling remained active at the time of surgery and whether remodeling was similar to previously reported remodeling observed in several animal models. Grossly, the hypertensive vessels appeared atherosclerotic. Histochemical stains revealed a thick, diffuse neointima in hypertensive vessels compared with normal vessels. Immunohistochemistry demonstrated elastin protein in the neointima and in situ hybridization studies demonstrated tropoelastin mRNA largely in the neointima. Similarly, immunohistochemistry and in situ hybridization detected cellular fibronectin, thrombospondin and type I collagen protein and mRNA within the thickened intima from hypertensive vessels. These studies provide evidence that hypertensive vessels in patients with severe chronic pulmonary hypertension are actively remodeling but that the pattern of remodeling is different from previously described animal models. Images Figure 1 Figure 2 Figure 3 PMID:1739129

  19. Regulatory effects of matrix protein variations on influenza virus growth.

    PubMed

    Yasuda, J; Toyoda, T; Nakayama, M; Ishihama, A

    1993-01-01

    Influenza virus A/WSN/33 forms large plaques (> 3 mm diameter) on MDCK cells whereas A/Aichi/2/68 forms only small plaques (< 1 mm diameter). Fast growing reassortants (AWM), isolated by mixed infection of MDCK cells with these two virus strains in the presence of anti-WSN antibodies, all carried the M gene from WSN. On MDCK cells, these reassortants produced progeny viruses as rapidly as did WSN, and the virus yield was as high as Aichi. The fast-growing reassortants overcame the growth inhibitory effect of lignins. Pulse-labeling experiments at various times after virus infection showed that the reassortant AWM started to synthesize viral proteins earlier than Aichi. Taken together, we conclude that upon infecting MDCK cells, the reassortant viruses advance rapidly into the growth cycle, thereby leading to an elevated level of progeny viruses in the early period of infection. Possible mechanisms of the M gene involvement in the determination of virus growth rate are discussed, in connection with multiple functions of the M proteins. PMID:8257290

  20. The incorporation of extracellular matrix proteins in protein polymer hydrogels to improve encapsulated beta-cell function.

    PubMed

    Beenken-Rothkopf, Liese N; Karfeld-Sulzer, Lindsay S; Davis, Nicolynn E; Forster, Ryan; Barron, Annelise E; Fontaine, Magali J

    2013-01-01

    Biomaterial encapsulation of islets has been proposed to improve the long-term success of islet transplantation by recreating a suitable microenvironment and enhancing cell-matrix interactions that affect cellular function. Protein polymer hydrogels previously showed promise as a biocompatible scaffold by maintaining high cell viability. Here, enzymatically-crosslinked protein polymers were used to investigate the effects of varying scaffold properties and of introducing ECM proteins on the viability and function of encapsulated MIN6 β-cells. Chemical and mechanical properties of the hydrogel were modified by altering the protein concentrations while collagen IV, fibronectin, and laminin were incorporated to reestablish cell-matrix interactions lost during cell isolation. Rheology indicated all hydrogels formed quickly, resulting in robust, elastic hydrogels with Young's moduli similar to soft tissue. All hydrogels tested supported both high MIN6 β-cell viability and function and have the potential to serve as an encapsulation platform for islet cell delivery in vivo.

  1. An acidic matrix protein, Pif, is a key macromolecule for nacre formation.

    PubMed

    Suzuki, Michio; Saruwatari, Kazuko; Kogure, Toshihiro; Yamamoto, Yuya; Nishimura, Tatsuya; Kato, Takashi; Nagasawa, Hiromichi

    2009-09-11

    The mollusk shell is a hard tissue consisting of calcium carbonate crystals and an organic matrix. The nacre of the shell is characterized by a stacked compartment structure with a uniformly oriented c axis of aragonite crystals in each compartment. Using a calcium carbonate-binding assay, we identified an acidic matrix protein, Pif, in the pearl oyster Pinctada fucata that specifically binds to aragonite crystals. The Pif complementary DNA (cDNA) encoded a precursor protein, which was posttranslationally cleaved to produce Pif 97 and Pif 80. The results from immunolocalization, a knockdown experiment that used RNA interference, and in vitro calcium carbonate crystallization studies strongly indicate that Pif regulates nacre formation.

  2. δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions.

    PubMed

    Yuan, Li; Seong, Eunju; Beuscher, James L; Arikkath, Jyothi

    2015-04-24

    The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome.

  3. δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions.

    PubMed

    Yuan, Li; Seong, Eunju; Beuscher, James L; Arikkath, Jyothi

    2015-04-24

    The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. PMID:25724647

  4. δ-Catenin Regulates Spine Architecture via Cadherin and PDZ-dependent Interactions*

    PubMed Central

    Yuan, Li; Seong, Eunju; Beuscher, James L.; Arikkath, Jyothi

    2015-01-01

    The ability of neurons to maintain spine architecture and modulate it in response to synaptic activity is a crucial component of the cellular machinery that underlies information storage in pyramidal neurons of the hippocampus. Here we show a critical role for δ-catenin, a component of the cadherin-catenin cell adhesion complex, in regulating spine head width and length in pyramidal neurons of the hippocampus. The loss of Ctnnd2, the gene encoding δ-catenin, has been associated with the intellectual disability observed in the cri du chat syndrome, suggesting that the functional roles of δ-catenin are vital for neuronal integrity and higher order functions. We demonstrate that loss of δ-catenin in a mouse model or knockdown of δ-catenin in pyramidal neurons compromises spine head width and length, without altering spine dynamics. This is accompanied by a reduction in the levels of synaptic N-cadherin. The ability of δ-catenin to modulate spine architecture is critically dependent on its ability to interact with cadherin and PDZ domain-containing proteins. We propose that loss of δ-catenin during development perturbs synaptic architecture leading to developmental aberrations in neural circuit formation that contribute to the learning disabilities in a mouse model and humans with cri du chat syndrome. PMID:25724647

  5. Rho protein GTPases and their interactions with NFκB: crossroads of inflammation and matrix biology

    PubMed Central

    Tong, Louis; Tergaonkar, Vinay

    2014-01-01

    The RhoGTPases, with RhoA, Cdc42 and Rac being major members, are a group of key ubiquitous proteins present in all eukaryotic organisms that subserve such important functions as cell migration, adhesion and differentiation. The NFκB (nuclear factor κB) is a family of constitutive and inducible transcription factors that through their diverse target genes, play a major role in processes such as cytokine expression, stress regulation, cell division and transformation. Research over the past decade has uncovered new molecular links between the RhoGTPases and the NFκB pathway, with the RhoGTPases playing a positive or negative regulatory role on NFκB activation depending on the context. The RhoA–NFκB interaction has been shown to be important in cytokine-activated NFκB processes, such as those induced by TNFα (tumour necrosis factor α). On the other hand, Rac is important for activating the NFκB response downstream of integrin activation, such as after phagocytosis. Specific residues of Rac1 are important for triggering NFκB activation, and mutations do obliterate this response. Other upstream triggers of the RhoGTPase–NFκB interactions include the suppressive p120 catenin, with implications for skin inflammation. The networks described here are not only important areas for further research, but are also significant for discovery of targets for translational medicine. PMID:24877606

  6. Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

    PubMed

    Dhore, C R; Cleutjens, J P; Lutgens, E; Cleutjens, K B; Geusens, P P; Kitslaar, P J; Tordoir, J H; Spronk, H M; Vermeer, C; Daemen, M J

    2001-12-01

    In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification. PMID:11742876

  7. 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

  8. An Overview of the Medical Applications of Marine Skeletal Matrix Proteins

    PubMed Central

    Rahman, M. Azizur

    2016-01-01

    In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery. PMID:27626432

  9. An Overview of the Medical Applications of Marine Skeletal Matrix Proteins.

    PubMed

    Rahman, M Azizur

    2016-01-01

    In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery. PMID:27626432

  10. An Overview of the Medical Applications of Marine Skeletal Matrix Proteins.

    PubMed

    Rahman, M Azizur

    2016-09-12

    In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.

  11. Measles Virus Matrix Protein Inhibits Host Cell Transcription.

    PubMed

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  12. Measles Virus Matrix Protein Inhibits Host Cell Transcription

    PubMed Central

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  13. Versatile Photocrosslinked Protein Hydrogel Matrix for Magnetic-Nanoparticle-Doping and Biomineralization.

    PubMed

    Ji, Fengying; Li, Shanpeng; Yang, Hai; Wang, Zhirui; Li, Aiwu

    2016-02-01

    A versatile template biomaterial was facilely obtained by ultraviolet (UV) photocrosslinking approach using protein molecules as building blocks. As-formed photocrosslinked protein hydrogel matrix (PPHM) was proved to be composed of covalently bound and dense packing protein molecules. Therefore, the PPHM was endowed with highly smooth topograghy with an average roughness of approximately 5 nm, and was self-supporting and flexible. The PPHM could be easily functionalized by doping Fe3O4 magnetic nanoparticles inside the protein hydrogel. Further, PPHM was experimentally demonstrated to be used as a applicable template for biomineralization. PMID:27433606

  14. Versatile Photocrosslinked Protein Hydrogel Matrix for Magnetic-Nanoparticle-Doping and Biomineralization.

    PubMed

    Ji, Fengying; Li, Shanpeng; Yang, Hai; Wang, Zhirui; Li, Aiwu

    2016-02-01

    A versatile template biomaterial was facilely obtained by ultraviolet (UV) photocrosslinking approach using protein molecules as building blocks. As-formed photocrosslinked protein hydrogel matrix (PPHM) was proved to be composed of covalently bound and dense packing protein molecules. Therefore, the PPHM was endowed with highly smooth topograghy with an average roughness of approximately 5 nm, and was self-supporting and flexible. The PPHM could be easily functionalized by doping Fe3O4 magnetic nanoparticles inside the protein hydrogel. Further, PPHM was experimentally demonstrated to be used as a applicable template for biomineralization.

  15. Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer

    PubMed Central

    Arighi, Cecilia N.; Wu, Cathy H.

    2015-01-01

    Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

  16. Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer.

    PubMed

    Çelen, İrem; Ross, Karen E; Arighi, Cecilia N; Wu, Cathy H

    2015-01-01

    Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge "maps" of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

  17. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    PubMed

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  18. IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Quirós, Miguel; Medina-Contreras, Oscar; Hamilton, Ross W.; Kolegraff, Keli N.; Koch, Stefan; Candelario, Aurora; Romo-Parra, Hector; Laur, Oskar; Hilgarth, Roland S.; Denning, Timothy L.; Parkos, Charles A.; Nusrat, Asma

    2014-01-01

    The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation. PMID:25079689

  19. Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix

    PubMed Central

    Garg, Sriram; Stölting, Jan; Zimorski, Verena; Rada, Petr; Tachezy, Jan; Martin, William F.; Gould, Sven B.

    2015-01-01

    The origin of protein import was a key step in the endosymbiotic acquisition of mitochondria. Though the main translocon of the mitochondrial outer membrane, TOM40, is ubiquitous among organelles of mitochondrial ancestry, the transit peptides, or N-terminal targeting sequences (NTSs), recognised by the TOM complex, are not. To better understand the nature of evolutionary conservation in mitochondrial protein import, we investigated the targeting behavior of Trichomonas vaginalis hydrogenosomal proteins in Saccharomyces cerevisiae and vice versa. Hydrogenosomes import yeast mitochondrial proteins even in the absence of their native NTSs, but do not import yeast cytosolic proteins. Conversely, yeast mitochondria import hydrogenosomal proteins with and without their short NTSs. Conservation of an NTS-independent mitochondrial import route from excavates to opisthokonts indicates its presence in the eukaryote common ancestor. Mitochondrial protein import is known to entail electrophoresis of positively charged NTSs across the electrochemical gradient of the inner mitochondrial membrane. Our present findings indicate that mitochondrial transit peptides, which readily arise from random sequences, were initially selected as a signal for charge-dependent protein targeting specifically to the mitochondrial matrix. Evolutionary loss of the electron transport chain in hydrogenosomes and mitosomes lifted the selective constraints that maintain positive charge in NTSs, allowing first the NTS charge, and subsequently the NTS itself, to be lost. This resulted in NTS-independent matrix targeting, which is conserved across the evolutionary divide separating trichomonads and yeast, and which we propose is the ancestral state of mitochondrial protein import. PMID:26338186

  20. Cloning, identification and functional analysis of a β-catenin homologue from Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Zhang, Shuang; Shi, Lili; L, Kai; Li, Haoyang; Wang, Sheng; He, Jianguo; Li, Chaozheng

    2016-07-01

    Wnt signaling is known to control multiple of cellular processes such as cell differentiation, communication, apoptosis and proliferation, and is also reported to play a role during microbial infection. β-catenin is a key regulator of the Wnt signaling cascade. In the present study, we cloned and identified a β-catenin homologue from Litopenaeus vannamei termed Lvβ-catenin. The full-length of Lvβ-catenin transcript was 2797 bp in length within a 2451 bp open reading frame (ORF) that encoded a protein of 816 amino acids. Lvβ-catenin protein was comprised of several characteristic domains such as an N-terminal region of GSK-β consensus phosphorylation site and Coed coil section, a central region of 12 continuous Armadillo/β-Catenin-like repeat (ARM) domains and a C-terminal region. Real-time PCR showed Lvβ-catenin expression was responsive to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Dual-reporter analysis showed that over-expression of Lvβ-catenin could induce activation of the promoter activities of several antimicrobial peptides (AMPs) such as shrimp PEN4, suggesting that Lvβ-catenin could play a role in regulating the production of AMPs. Knockdown of Lvβ-catenin enhanced the sensitivity of shrimps to V. parahaemolyticus and WSSV challenge, suggesting Lvβ-catenin could play a positive role against bacterial and viral pathogens. In summary, the results presented in this study provided some insights into the function of Wnt/β-catenin of shrimp in regulating AMPs and the host defense against invading pathogens. PMID:27036405

  1. Depletion of γ-catenin by Histone Deacetylase Inhibition Confers Elimination of CML Stem Cells in Combination with Imatinib.

    PubMed

    Jin, Yanli; Yao, Yiwu; Chen, Li; Zhu, Xiaohui; Jin, Bei; Shen, Yingying; Li, Juan; Du, Xin; Lu, Yuhong; Jiang, Sheng; Pan, Jingxuan

    2016-01-01

    Quiescent leukemia stem cells (LSCs) that are insensitive to BCR-ABL tyrosine kinase inhibitors confer resistance to imatinib in chronic myelogenous leukemia (CML). Identifying proteins to regulate survival and stemness of LSCs is urgently needed. Although histone deacetylase inhibitors (HDACis) can eliminate quiescent LSCs in CML, little is known about the underlying mechanism that HDACis kill LSCs. By fishing with a biotin-labeled probe, we identified that HDACi JSL-1 bound to the protein γ-catenin. γ-Catenin expression was higher in LSCs from CML patients than normal hematopoietic stem cells. Silencing γ-catenin in human CML CD34(+) bone-marrow (BM) cells sufficiently eliminated LSCs, which suggests that γ-catenin is required for survival of CML LSCs. Pharmacological inhibition of γ-catenin thwarted survival and self-renewal of human CML CD34(+) cells in vitro, and of murine LSCs in BCR-ABL-driven CML mice. γ-Catenin inhibition reduced long-term engraftment of human CML CD34(+) cells in NOD.Cg-Prkdc (scid) II2rg (tm1Sug)/JicCrl (NOG) mice. Silencing γ-catenin by shRNA in human primary CD34(+) cells did not alter β-catenin, implying a β-catenin-independent role of γ-catenin in survival and self-renewal of CML LSCs. Taken together, our findings validate that γ-catenin may be a novel therapeutic target of LSCs, and suppression of γ-catenin by HDACi may explain elimination of CML LSCs. PMID:27570562

  2. Depletion of γ-catenin by Histone Deacetylase Inhibition Confers Elimination of CML Stem Cells in Combination with Imatinib

    PubMed Central

    Jin, Yanli; Yao, Yiwu; Chen, Li; Zhu, Xiaohui; Jin, Bei; Shen, Yingying; Li, Juan; Du, Xin; Lu, Yuhong; Jiang, Sheng; Pan, Jingxuan

    2016-01-01

    Quiescent leukemia stem cells (LSCs) that are insensitive to BCR-ABL tyrosine kinase inhibitors confer resistance to imatinib in chronic myelogenous leukemia (CML). Identifying proteins to regulate survival and stemness of LSCs is urgently needed. Although histone deacetylase inhibitors (HDACis) can eliminate quiescent LSCs in CML, little is known about the underlying mechanism that HDACis kill LSCs. By fishing with a biotin-labeled probe, we identified that HDACi JSL-1 bound to the protein γ-catenin. γ-Catenin expression was higher in LSCs from CML patients than normal hematopoietic stem cells. Silencing γ-catenin in human CML CD34+ bone-marrow (BM) cells sufficiently eliminated LSCs, which suggests that γ-catenin is required for survival of CML LSCs. Pharmacological inhibition of γ-catenin thwarted survival and self-renewal of human CML CD34+ cells in vitro, and of murine LSCs in BCR-ABL-driven CML mice. γ-Catenin inhibition reduced long-term engraftment of human CML CD34+ cells in NOD.Cg-Prkdcscid II2rgtm1Sug/JicCrl (NOG) mice. Silencing γ-catenin by shRNA in human primary CD34+ cells did not alter β-catenin, implying a β-catenin-independent role of γ-catenin in survival and self-renewal of CML LSCs. Taken together, our findings validate that γ-catenin may be a novel therapeutic target of LSCs, and suppression of γ-catenin by HDACi may explain elimination of CML LSCs. PMID:27570562

  3. Cloning, identification and functional analysis of a β-catenin homologue from Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Zhang, Shuang; Shi, Lili; L, Kai; Li, Haoyang; Wang, Sheng; He, Jianguo; Li, Chaozheng

    2016-07-01

    Wnt signaling is known to control multiple of cellular processes such as cell differentiation, communication, apoptosis and proliferation, and is also reported to play a role during microbial infection. β-catenin is a key regulator of the Wnt signaling cascade. In the present study, we cloned and identified a β-catenin homologue from Litopenaeus vannamei termed Lvβ-catenin. The full-length of Lvβ-catenin transcript was 2797 bp in length within a 2451 bp open reading frame (ORF) that encoded a protein of 816 amino acids. Lvβ-catenin protein was comprised of several characteristic domains such as an N-terminal region of GSK-β consensus phosphorylation site and Coed coil section, a central region of 12 continuous Armadillo/β-Catenin-like repeat (ARM) domains and a C-terminal region. Real-time PCR showed Lvβ-catenin expression was responsive to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infection. Dual-reporter analysis showed that over-expression of Lvβ-catenin could induce activation of the promoter activities of several antimicrobial peptides (AMPs) such as shrimp PEN4, suggesting that Lvβ-catenin could play a role in regulating the production of AMPs. Knockdown of Lvβ-catenin enhanced the sensitivity of shrimps to V. parahaemolyticus and WSSV challenge, suggesting Lvβ-catenin could play a positive role against bacterial and viral pathogens. In summary, the results presented in this study provided some insights into the function of Wnt/β-catenin of shrimp in regulating AMPs and the host defense against invading pathogens.

  4. Culture of outer epithelial cells from mantle tissue to study shell matrix protein secretion for biomineralization.

    PubMed

    Gong, Ningping; Li, Qi; Huang, Jing; Fang, Zi; Zhang, Guiyou; Xie, Liping; Zhang, Rongqing

    2008-09-01

    Mantle tissue plays an important role in shell biomineralization by secreting matrix proteins for shell formation. However, the mechanism by which it regulates matrix protein secretion is poorly understood, largely because of the lack of cellular tools for in vitro study and techniques to evaluate matrix protein secretion. We have isolated the outer epithelial cells of the mantle of the pearl oyster, Pinctada fucata, and evaluated cellular metabolism by measuring the secretion of the matrix protein, nacrein. A novel sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was established to quantify nacrein. Mantle explant culture was demonstrated to provide dissociated tissue cells with high viability. Single dissociated cell types from explant culture were separated by density in a discontinuous Percoll gradient. The outer epithelial cells were isolated from other cell types by their higher density and identified by immunolabeling and ultrastructure analysis. ELISA assays revealed that the outer epithelial cells retained the ability to secrete nacrein in vitro. Moreover, increased nacrein secretion resulted from an increased Ca(2+) concentration in the culture media of the outer epithelial cells, in a concentration-dependent manner. These results confirm that outer epithelial cell culture and the ELISA method are useful tools for studying the regulatory mechanisms of shell biomineralization.

  5. Extracellular Matrix Protein-Coated Scaffolds Promote the Reversal of Diabetes After Extrahepatic Islet Transplantation

    PubMed Central

    Salvay, David M.; Rives, Christopher B.; Zhang, Xiaomin; Chen, Fei; Kaufman, Dixon B.; Lowe, William L.; Shea, Lonnie D.

    2008-01-01

    Background The survival and function of transplanted pancreatic islets is limited, owing in part to disruption of islet-matrix attachments during the isolation procedure. Using polymer scaffolds as a platform for islet transplantation, we investigated the hypothesis that replacement of key extracellular matrix components known to surround islets in vivo would improve graft function at an extrahepatic implantation site. Methods Microporous polymer scaffolds fabricated from copolymers of lactide and glycolide were adsorbed with collagen IV, fibronectin, laminin-332 or serum proteins before seeding with 125 mouse islets. Islet-seeded scaffolds were then implanted onto the epididymal fat pad of syngeneic mice with streptozotocin-induced diabetes. Nonfasting glucose levels, weight gain, response to glucose challenges, and histology were used to assess graft function for 10 months after transplantation. Results Mice transplanted with islets seeded onto scaffolds adsorbed with collagen IV achieved euglycemia fastest and their response to glucose challenge was similar to normal mice. Fibronectin and laminin similarly promoted euglycemia, yet required more time than collagen IV and less time than serum. Histopathological assessment of retrieved grafts demonstrated that coating scaffolds with specific extracellular matrix proteins increased total islet area in the sections and vessel density within the transplanted islets, relative to controls. Conclusions Extracellular matrix proteins adsorbed to microporous scaffolds can enhance the function of transplanted islets, with collagen IV maximizing graft function relative to the other proteins tested. These scaffolds enable the creation of well-defined microenvironments that promote graft efficacy at extrahepatic sites. PMID:18497687

  6. Osteopontin induces {beta}-catenin signaling through activation of Akt in prostate cancer cells

    SciTech Connect

    Robertson, Brian W.; Chellaiah, Meenakshi A.

    2010-01-01

    Secretion of osteopontin (OPN) by cancer cells is a known mediator of tumorigenesis and cancer progression in both experimental and clinical studies. Our work demonstrates that OPN can activate Akt, an important step in cancer progression. Both ILK and PI3K are integral proteins in the OPN/Akt pathway, as inhibition of either kinase leads to a loss of OPN-mediated Akt activation. Subsequent to OPN-induced Akt activation, we observe inactivation of GSK-3{beta}, a regulator of {beta}-catenin. Osteopontin stimulation leads to an overall increase in {beta}-catenin protein levels with a resultant transfer of {beta}-catenin to the nucleus. Through the nuclear import of {beta}-catenin, OPN increases both the transcription and protein levels of MMP-7 and CD44, which are known TCF/LEF transcription targets. This work describes an important aspect of cancer progression induced by OPN.

  7. SOX9 Regulates Low Density Lipoprotein Receptor-related Protein 6 (LRP6) and T-cell Factor 4 (TCF4) Expression and Wnt/β-catenin Activation in Breast Cancer*

    PubMed Central

    Wang, Hongyun; He, Lingfeng; Ma, Fen; Regan, Meredith M.; Balk, Steven P.; Richardson, Andrea L.; Yuan, Xin

    2013-01-01

    Gene expression profiling has identified breast cancer (BCa) subtypes, including an aggressive basal-like (BL) subtype. The molecular signals underlying the behavior observed in BL-BCa group are largely unknown, although recent results indicate a prevalent increase in Wnt/β-catenin activity. Our immunohistochemistry study confirmed that SOX9, one of the BL-BCa signature genes, was expressed by most BL-BCa, and its expression correlated with indicators of poor prognosis. Importantly, BCa gene expression profiling strongly associated SOX9 with the expression of Wnt/β-catenin pathway components, LRP6 and TCF4. In cancer cell lines, SOX9 silencing reduced cell proliferation and invasion, LRP6 and TCF4 transcription, and decreased Wnt/β-catenin activation. SOX9 expression was also increased by Wnt, indicating that SOX9 is at the center of a positive feedback loop that enhances Wnt/β-catenin signaling. Consistently, SOX9 overexpression in BCa cell lines and transgenic SOX9 expression in breast epithelium caused increased LRP6 and TCF4 expression and Wnt/β-catenin activation. These results identify SOX9-mediated Wnt/β-catenin activation as one of the molecular mechanisms underlying aberrant Wnt/β-catenin activity in BCa, especially in the BL-BCa subgroup. PMID:23306204

  8. Activation of β-catenin Signaling by TFF1 Loss Promotes Cell Proliferation and Gastric Tumorigenesis

    PubMed Central

    Soutto, Mohammed; Peng, DunFa; Katsha, Ahmed; Chen, Zheng; Piazuelo, M. Blanca; Washington, M. Kay; Belkhiri, Abbes; Correa, Pelayo; El-Rifai, Wael

    2014-01-01

    Objective In this study, we investigated the role of TFF1 in regulating cell proliferation and tumor development through β-catenin signaling using in vivo and in vitro models of gastric tumorigenesis. Design TFF1-Knockout mice, Immunohistochemistry, luciferase reporter, qRT-PCR, immunoblot and phosphatase assays were used to examine the role of TFF1 on β-catenin signaling pathway. Results Nuclear localization of β-catenin with transcriptional upregulation of its target genes, c-Myc and Ccnd1, was detected in hyperplastic tissue at early age of 4-6 weeks and maintained during all stages of gastric tumorigenesis in the Tff1-knockout mice. The reconstitution of TFF1 or TFF1 conditioned media significantly inhibited the β-catenin/TCF transcription activity in MKN28 gastric cancer cells. In agreement with these results, we detected a reduction in the levels of nuclear β-catenin with downregulation of c-MYC and CCND1 mRNA. Analysis of signaling molecules upstream of β-catenin revealed a decrease in p-GSK3β (Ser9) and p-AKT (Ser473) protein levels following the reconstitution of TFF1 expression; this was consistent with the increase of p-β-catenin (Ser33/37/Thr41) and decrease of p-β-catenin (Ser552). This TFF1-induced reduction in phosphorylation of GSK3β and AKT was dependent on PP2A activity. The treatment with okadaic acid or knockdown of PP2A abrogated these effects. Consistent with the mouse data, we observed loss of TFF1 and an increase in nuclear localization of β-catenin in stages of human gastric tumorigenesis. Conclusion Our data indicate that loss of TFF1 promotes β-catenin activation and gastric tumorigenesis through regulation of PP2A, a major regulator of AKT-GSK3β signaling. PMID:25107557

  9. Pleiotrophin induces neurite outgrowth and up-regulates growth-associated protein (GAP)-43 mRNA through the ALK/GSK3beta/beta-catenin signaling in developing mouse neurons.

    PubMed

    Yanagisawa, Hiroko; Komuta, Yukari; Kawano, Hitoshi; Toyoda, Masashi; Sango, Kazunori

    2010-01-01

    Pleiotrophin (PTN) is highly expressed in the nervous system during embryogenesis; however, little is known about its functional role in neural development. By using whole mount in situ hybridization, we observed that the expression pattern of PTN was similar to that of Wnt3a; PTN mRNA was abundant in the nervous tissue along the dorsal midline and in the forelimb and hindlimb buds of embryonic mice (E8.5-E12.5). Treatment with recombinant PTN (100ng/ml) induced phosphorylation of glycogen synthase kinase 3beta (GSK3beta), nuclear localization of beta-catenin and up-regulation of growth-associated protein (GAP)-43 mRNA in cultured embryonic mouse (E14.5) neurons. Furthermore, recombinant PTN enhanced neurite outgrowth from cortical explants embedded in Matrigel. These PTN-induced biochemical changes and neurite outgrowth were attenuated by the co-treatment with anti-anaplastic lymphoma kinase (ALK) antibodies, but not with anti-protein tyrosine phosphatase (PTP)zeta antibodies. These findings imply that ALK is involved in the PTN signaling on neural development.

  10. 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.

  11. R-ETODOLAC DECREASES BETA-CATENIN LEVELS ALONG WITH SURVIVAL AND PROLIFERATION OF HEPATOMA CELLS

    PubMed Central

    Behari, Jaideep; Zeng, Gang; Otruba, Wade; Thompson, Michael; Muller, Peggy; Micsenyi, Amanda; Sekhon, Sandeep S.; Leoni, Lorenzo; Monga, Satdarshan P. S.

    2007-01-01

    Background Inhibition of hepatoma cells by cyclooxygenase (COX)-2 dependent and independent mechanisms has been shown previously. Here, we examine the effect of Celecoxib, a COX-2-inhibitor and R-Etodolac, an enantiomer of the nonsteroidal anti-inflammatory drug Etodolac, which lacks COX-inhibitory activity, on the Wnt/β-catenin pathway and human hepatoma cells. Methods Hep3B and HepG2 cell lines were treated with Celecoxib or R-Etodolac, and examined for viability, DNA synthesis, Wnt/β-catenin pathway components, and downstream target gene expression. Results Celecoxib at high doses affected β-catenin protein by inducing its degradation via GSK3β and APC along with diminished tumor cell proliferation and survival. R-Etodolac at physiological doses caused decrease in total and activated β-catenin protein secondary to decrease in its gene expression and post-translationally through GSK3β activation. In addition, increased β-catenin-E-cadherin was also observed at the membrane. An associated inhibition of β-catenin-dependent Tcf reporter activity, decreased levels of downstream target gene products glutamine synthetase and cyclin-D1, and decreased proliferation and survival of hepatoma cells was evident. Conclusion The antitumor effects of Celecoxib (at high concentrations) and R-Etodolac (at physiological doses) on HCC cells were accompanied by the down-regulation of β-catenin demonstrating a useful therapeutic strategy in hepatocellular cancer. PMID:17275129

  12. Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.

    PubMed

    Mass, Tali; Drake, Jeana L; Peters, Esther C; Jiang, Wenge; Falkowski, Paul G

    2014-09-01

    The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral's metabolic pathways.

  13. Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata

    PubMed Central

    Mass, Tali; Drake, Jeana L.; Peters, Esther C.; Jiang, Wenge; Falkowski, Paul G.

    2014-01-01

    The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral’s metabolic pathways. PMID:25139990

  14. Pam17 and Tim44 act sequentially in protein import into the mitochondrial matrix.

    PubMed

    Schiller, Dirk

    2009-11-01

    Import of proteins into the matrix is driven by the Tim23 presequence translocase-associated import motor PAM. The core component of PAM is the mitochondrial chaperone mtHsp70, which ensures efficient translocation of proteins across the inner membrane through interactions with the J-protein complex Pam16-Pam18 (Tim16-Tim14) and its cochaperone Tim44. The recently identified non-essential Pam17 is a further member of PAM. Genetic and biochemical analyses reveal synthetic interactions between PAM17 and TIM44. Pam17 is involved in an early stage of protein translocation whereas Tim44 assists in a later step of transport, suggesting that both proteins can cooperate in a complementary manner in protein import.

  15. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  16. Argon Laser Welding Induces Degradation And Crosslinking Of Extracellular Matrix Protein: A Preliminary Report

    NASA Astrophysics Data System (ADS)

    Su, Lyndon; Murray, Louann W.; White, Rodney A.; Kopchok, George E.; Guthrie, Carol; White, Geoffrey H.

    1989-09-01

    Extracellular matrix components from untreated and laser-welded skin and blood vessels were extracted with guanidine hydrochloride and separated by SDS polyacrylamide gel electrophoresis. When compared to matched, untreated tissues, protein electrophoretic profiles from laser-treated samples showed several changes. In both tissue types, the concentration of a protein normally migrating between alpha and beta chains of type I collagen decreased in laser-treated samples. However, laser-treated blood vessels showed significantly more low molecular weight protein, whereas significantly more high molecular weight protein appeared in laser-treated skin samples when compared to untreated tissue. These results suggest that the argon laser either degrades or crosslinks proteins in-vivo. Laser induced protein crosslinks may be the biochemical basis of laser welding.

  17. Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins.

    PubMed Central

    Nooteboom, A; Hendriks, T; Ottehöller, I; van der Linden, C J

    2000-01-01

    OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators. PMID:11200364

  18. A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

    PubMed Central

    Gonzalez, Nicola H.; Felsner, Gregor; Schramm, Frederic D.; Klingl, Andreas; Maier, Uwe-G.; Bolte, Kathrin

    2011-01-01

    Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1. PMID:21966495

  19. The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae.

    PubMed

    Johnson, Tanya L; Fong, Jiunn C; Rule, Chelsea; Rogers, Andrew; Yildiz, Fitnat H; Sandkvist, Maria

    2014-12-01

    Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥ 20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies. PMID:25266381

  20. Matrix Gla Protein Binds to Fibronectin and Enhances Cell Attachment and Spreading on Fibronectin

    PubMed Central

    Nishimoto, Satoru Ken; Nishimoto, Miyako

    2014-01-01

    Background. Matrix Gla protein (MGP) is a vitamin K-dependent, extracellular matrix protein. MGP is a calcification inhibitor of arteries and cartilage. However MGP is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells. The presence of MGP in those instances does not correlate well with the calcification inhibitory role. This study explores a potential mechanism for MGP to bind to matrix proteins and alter cell matrix interactions. Methods. To determine whether MGP influences cell behavior through interaction with fibronectin, we studied MGP binding to fibronectin, the effect of MGP on fibronectin mediated cell attachment and spreading and immunolocalized MGP and fibronectin. Results. First, MGP binds to fibronectin. The binding site for MGP is in a specific fibronectin fragment, called III1-C or anastellin. The binding site for fibronectin is in a MGP C-terminal peptide comprising amino acids 61–77. Second, MGP enhances cell attachment and cell spreading on fibronectin. MGP alone does not promote cell adhesion. Third, MGP is present in fibronectin-rich regions of tissue sections. Conclusions. MGP binds to fibronectin. The presence of MGP increased cell-fibronectin interactions. PMID:25210519

  1. OLIGOMERIZATION OF A RETROVIRAL MATRIX PROTEIN IS FACILITATED BY BACKBONE FLEXIBILITY ON NS TIMESCALE

    PubMed Central

    Srb, Pavel; Vlach, Jiří; Prchal, Jan; Grocký, Marián; Ruml, Tomáš; Lang, Jan; Hrabal, Richard

    2011-01-01

    Oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidences have been obtained that the oligomerization capacity of the wild type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on ns timescale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural pre-organization play an important role in the control of the oligomerization process. PMID:21366213

  2. Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein.

    PubMed

    McPhee, Helen K; Carlisle, Jennifer L; Beeby, Andrew; Money, Victoria A; Watson, Scott M D; Yeo, R Paul; Sanderson, John M

    2011-01-01

    The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. In ternary mixtures composed of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Prolonged incubation of the protein with mixtures of phosphocholines and phosphoethanolamines leads to the formation of helical protein assemblies of uniform diameter that demonstrate an inherent propensity of the protein to assemble into a filamentous form. PMID:21141948

  3. Nipah Virus Matrix Protein Influences Fusogenicity and Is Essential for Particle Infectivity and Stability

    PubMed Central

    Dietzel, Erik; Kolesnikova, Larissa; Sawatsky, Bevan; Heiner, Anja; Weis, Michael; Kobinger, Gary P.; Becker, Stephan; von Messling, Veronika

    2015-01-01

    ABSTRACT Nipah virus (NiV) causes fatal encephalitic infections in humans. To characterize the role of the matrix (M) protein in the viral life cycle, we generated a reverse genetics system based on NiV strain Malaysia. Using an enhanced green fluorescent protein (eGFP)-expressing M protein-deleted NiV, we observed a slightly increased cell-cell fusion, slow replication kinetics, and significantly reduced peak titers compared to the parental virus. While increased amounts of viral proteins were found in the supernatant of cells infected with M-deleted NiV, the infectivity-to-particle ratio was more than 100-fold reduced, and the particles were less thermostable and of more irregular morphology. Taken together, our data demonstrate that the M protein is not absolutely required for the production of cell-free NiV but is necessary for proper assembly and release of stable infectious NiV particles. IMPORTANCE Henipaviruses cause a severe disease with high mortality in human patients. Therefore, these viruses can be studied only in biosafety level 4 (BSL-4) laboratories, making it more challenging to characterize their life cycle. Here we investigated the role of the Nipah virus matrix protein in virus-mediated cell-cell fusion and in the formation and release of newly produced particles. We found that even though low levels of infectious viruses are produced in the absence of the matrix protein, it is required for the release of highly infectious and stable particles. Fusogenicity of matrixless viruses was slightly enhanced, further demonstrating the critical role of this protein in different steps of Nipah virus spread. PMID:26676785

  4. Identification of Protein-Protein Interactions via a Novel Matrix-Based Sequence Representation Model with Amino Acid Contact Information.

    PubMed

    Ding, Yijie; Tang, Jijun; Guo, Fei

    2016-09-24

    Identification of protein-protein interactions (PPIs) is a difficult and important problem in biology. Since experimental methods for predicting PPIs are both expensive and time-consuming, many computational methods have been developed to predict PPIs and interaction networks, which can be used to complement experimental approaches. However, these methods have limitations to overcome. They need a large number of homology proteins or literature to be applied in their method. In this paper, we propose a novel matrix-based protein sequence representation approach to predict PPIs, using an ensemble learning method for classification. We construct the matrix of Amino Acid Contact (AAC), based on the statistical analysis of residue-pairing frequencies in a database of 6323 protein-protein complexes. We first represent the protein sequence as a Substitution Matrix Representation (SMR) matrix. Then, the feature vector is extracted by applying algorithms of Histogram of Oriented Gradient (HOG) and Singular Value Decomposition (SVD) on the SMR matrix. Finally, we feed the feature vector into a Random Forest (RF) for judging interaction pairs and non-interaction pairs. Our method is applied to several PPI datasets to evaluate its performance. On the S . c e r e v i s i a e dataset, our method achieves 94 . 83 % accuracy and 92 . 40 % sensitivity. Compared with existing methods, and the accuracy of our method is increased by 0 . 11 percentage points. On the H . p y l o r i dataset, our method achieves 89 . 06 % accuracy and 88 . 15 % sensitivity, the accuracy of our method is increased by 0 . 76 % . On the H u m a n PPI dataset, our method achieves 97 . 60 % accuracy and 96 . 37 % sensitivity, and the accuracy of our method is increased by 1 . 30 % . In addition, we test our method on a very important PPI network, and it achieves 92 . 71 % accuracy. In the Wnt-related network, the accuracy of our method is increased by 16 . 67 % . The source code and all datasets are available

  5. Identification of Protein-Protein Interactions via a Novel Matrix-Based Sequence Representation Model with Amino Acid Contact Information.

    PubMed

    Ding, Yijie; Tang, Jijun; Guo, Fei

    2016-01-01

    Identification of protein-protein interactions (PPIs) is a difficult and important problem in biology. Since experimental methods for predicting PPIs are both expensive and time-consuming, many computational methods have been developed to predict PPIs and interaction networks, which can be used to complement experimental approaches. However, these methods have limitations to overcome. They need a large number of homology proteins or literature to be applied in their method. In this paper, we propose a novel matrix-based protein sequence representation approach to predict PPIs, using an ensemble learning method for classification. We construct the matrix of Amino Acid Contact (AAC), based on the statistical analysis of residue-pairing frequencies in a database of 6323 protein-protein complexes. We first represent the protein sequence as a Substitution Matrix Representation (SMR) matrix. Then, the feature vector is extracted by applying algorithms of Histogram of Oriented Gradient (HOG) and Singular Value Decomposition (SVD) on the SMR matrix. Finally, we feed the feature vector into a Random Forest (RF) for judging interaction pairs and non-interaction pairs. Our method is applied to several PPI datasets to evaluate its performance. On the S . c e r e v i s i a e dataset, our method achieves 94 . 83 % accuracy and 92 . 40 % sensitivity. Compared with existing methods, and the accuracy of our method is increased by 0 . 11 percentage points. On the H . p y l o r i dataset, our method achieves 89 . 06 % accuracy and 88 . 15 % sensitivity, the accuracy of our method is increased by 0 . 76 % . On the H u m a n PPI dataset, our method achieves 97 . 60 % accuracy and 96 . 37 % sensitivity, and the accuracy of our method is increased by 1 . 30 % . In addition, we test our method on a very important PPI network, and it achieves 92 . 71 % accuracy. In the Wnt-related network, the accuracy of our method is increased by 16 . 67 % . The source code and all datasets are available

  6. Sustained Wnt/β-catenin signalling causes neuroepithelial aberrations through the accumulation of aPKC at the apical pole.

    PubMed

    Herrera, Antonio; Saade, Murielle; Menendez, Anghara; Marti, Elisa; Pons, Sebastian

    2014-01-01

    β-Catenin mediates the canonical Wnt pathway by stimulating Tcf-dependent transcription and also associates to N-cadherin at the apical complex (AC) of neuroblasts. Here, we show that while β-catenin activity is required to form the AC and to maintain the cell polarity, oncogenic mutations that render stable forms of β-catenin (sβ-catenin) maintain the stemness of neuroblasts, inhibiting their differentiation and provoking aberrant growth. In examining the transcriptional and structural roles of β-catenin, we find that while β-catenin/Tcf transcriptional activity induces atypical protein kinase C (aPKC) expression, an alternative effect of β-catenin restricts aPKC to the apical pole of neuroepithelial cells. In agreement, we show that a constitutively active form of aPKC reproduces the neuroepithelial aberrations induced by β-catenin. Therefore, we conclude that β-catenin controls the cell fate and polarity of the neuroblasts through the expression and localization of aPKC. PMID:24942669

  7. 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.

  8. A Model Sea Urchin Spicule Matrix Protein Self-Associates To Form Mineral-Modifying Protein Hydrogels.

    PubMed

    Jain, Gaurav; Pendola, Martin; Rao, Ashit; Cölfen, Helmut; Evans, John Spencer

    2016-08-01

    In the purple sea urchin Strongylocentrotus purpuratus, the formation and mineralization of fracture-resistant skeletal elements such as the embryonic spicule require the combinatorial participation of numerous spicule matrix proteins such as the SpSM30A-F isoforms. However, because of limited abundance, it has been difficult to pursue extensive biochemical studies of the SpSM30 proteins and deduce their role in spicule formation and mineralization. To circumvent these problems, we expressed a model recombinant spicule matrix protein, rSpSM30B/C, which possesses the key sequence attributes of isoforms "B" and "C". Our findings indicate that rSpSM30B/C is expressed in insect cells as a single polypeptide containing variations in glycosylation that create microheterogeneity in rSpSM30B/C molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic mono- and bisialylated and mono- and bisulfated monosaccharides on the protein molecules and enhance its aggregation propensity. Bioinformatics and biophysical experiments confirm that rSpSM30B/C is an intrinsically disordered, aggregation-prone protein that forms porous protein hydrogels that control the in vitro mineralization process in three ways: (1) increase the time interval for prenucleation cluster formation and transiently stabilize an ACC polymorph, (2) promote and organize single-crystal calcite nanoparticles, and (3) promote faceted growth and create surface texturing of calcite crystals. These features are also common to mollusk shell nacre proteins, and we conclude that rSpSM30B/C is a spiculogenesis protein that exhibits traits found in other calcium carbonate mineral modification proteins. PMID:27426695

  9. In-depth proteomic analysis of shell matrix proteins of Pinctada fucata.

    PubMed

    Liu, Chuang; Li, Shiguo; Kong, Jingjing; Liu, Yangjia; Wang, Tianpeng; Xie, Liping; Zhang, Rongqing

    2015-11-26

    The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

  10. In-depth proteomic analysis of shell matrix proteins of Pinctada fucata

    PubMed Central

    Liu, Chuang; Li, Shiguo; Kong, Jingjing; Liu, Yangjia; Wang, Tianpeng; Xie, Liping; Zhang, Rongqing

    2015-01-01

    The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment. PMID:26608573

  11. Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo.

    PubMed Central

    Black, B L; Lyles, D S

    1992-01-01

    Infection by vesicular stomatitis virus (VSV) results in a rapid inhibition of host cell transcription and translation. To determine whether the viral matrix (M) protein was involved in this inhibition of host cell gene expression, an M protein expression vector was cotransfected with a target gene vector, encoding the target gene, encoding chloramphenicol acetyltransferase (CAT). Expression of M protein caused a decrease in CAT activity in a gene dosage-dependent manner, and inhibition was apparent by 12 h posttransfection. The inhibitory effect of M protein was quite potent. The level of M protein required for a 10-fold inhibition of CAT activity was less than 1% of the level of M protein produced during the sixth hour of VSV infection. Northern (RNA) analysis of cotransfected cells showed that expression of M protein caused a reduction in the steady-state level of the vector-encoded mRNAs. Expression of both CAT and M mRNAs was reduced in cells cotransfected with a plasmid encoding M protein, indicating that expression of small amounts of M protein from plasmid DNA inhibits further expression of both M and CAT mRNAs. Nuclear runoff transcription analysis demonstrated that expression of M protein inhibited transcription of the target genes. This is the first report of a viral gene product which is capable of inhibiting transcription in vivo in the absence of any other viral component. Images PMID:1318397

  12. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

  13. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics. PMID:27314233

  14. CHD8 Is an ATP-Dependent Chromatin Remodeling Factor That Regulates β-Catenin Target Genes▿

    PubMed Central

    Thompson, Brandi A.; Tremblay, Véronique; Lin, Grace; Bochar, Daniel A.

    2008-01-01

    ATP-dependent chromatin remodeling by the CHD family of proteins plays an important role in the regulation of gene transcription. Here we report that full-length CHD8 interacts directly with β-catenin and that CHD8 is also recruited specifically to the promoter regions of several β-catenin-responsive genes. Our results indicate that CHD8 negatively regulates β-catenin-targeted gene expression, since short hairpin RNA against CHD8 results in the activation of several β-catenin target genes. This regulation is also conserved through evolution; RNA interference against kismet, the apparent Drosophila ortholog of CHD8, results in a similar activation of β-catenin target genes. We also report the first demonstration of chromatin remodeling activity for a member of the CHD6-9 family of proteins, suggesting that CHD8 functions in transcription through the ATP-dependent modulation of chromatin structure. PMID:18378692

  15. Genetic alterations of δ-catenin/NPRAP/Neurojungin (CTNND2): functional implications in complex human diseases.

    PubMed

    Lu, Qun; Aguilar, Byron J; Li, Mingchuan; Jiang, Yongguang; Chen, Yan-Hua

    2016-10-01

    Some genes involved in complex human diseases are particularly vulnerable to genetic variations such as single nucleotide polymorphism, copy number variations, and mutations. For example, Ras mutations account for over 30 % of all human cancers. Additionally, there are some genes that can display different variations with functional impact in different diseases that are unrelated. One such gene stands out: δ-catenin/NPRAP/Neurojungin with gene designation as CTNND2 on chromosome 5p15.2. Recent advances in genome wide association as well as molecular biology approaches have uncovered striking involvement of δ-catenin gene variations linked to complex human disorders. These disorders include cancer, bipolar disorder, schizophrenia, autism, Cri-du-chat syndrome, myopia, cortical cataract-linked Alzheimer's disease, and infectious diseases. This list has rapidly grown longer in recent years, underscoring the pivotal roles of δ-catenin in critical human diseases. δ-Catenin is an adhesive junction-associated protein in the delta subfamily of the β-catenin superfamily. δ-Catenin functions in Wnt signaling to regulate gene expression and modulate Rho GTPases of the Ras superfamily in cytoskeletal reorganization. δ-Catenin likely lies where Wnt signaling meets Rho GTPases and is a unique and vulnerable common target for mutagenesis in different human diseases.

  16. Generation and behavioral characterization of beta-catenin forebrain-specific conditional knock-out mice.

    PubMed

    Gould, Todd D; O'Donnell, Kelley C; Picchini, Alyssa M; Dow, Eliot R; Chen, Guang; Manji, Husseini K

    2008-05-16

    The canonical Wnt pathway and beta-catenin have been implicated in the pathophysiology of mood disorders. We generated forebrain-specific CRE-mediated conditional beta-catenin knock-out mice to begin exploring the behavioral implications of decreased Wnt pathway signaling in the central nervous system. In situ hybridization revealed a progressive knock-out of beta-catenin that began between 2 and 4 weeks of age, and by 12 weeks resulted in considerably decreased beta-catenin expression in regions of the forebrain, including the frontal cortex, hippocampus, and striatum. A significant decrease in protein levels of beta-catenin in these brain regions was observed by Western blot. Behavioral characterization of these mice in several tests (including the forced swim test, tail suspension test (TST), learned helplessness, response and sensitization to stimulants, and light/dark box among other tests) revealed relatively circumscribed alterations. In the TST, knock-out mice spent significantly less time struggling (a depression-like phenotype). However, knock-out mice did not differ from their wild-type littermates in the other behavioral tests of mood-related or anxiety-related behaviors. These results suggest that a 60-70% beta-catenin reduction in circumscribed brain regions is only capable of inducing subtle behavioral changes. Alternatively, regulating beta-catenin may modulate drug effects rather than being a model of mood disorder pathophysiology per se.

  17. Genetic alterations of δ-catenin/NPRAP/Neurojungin (CTNND2): functional implications in complex human diseases.

    PubMed

    Lu, Qun; Aguilar, Byron J; Li, Mingchuan; Jiang, Yongguang; Chen, Yan-Hua

    2016-10-01

    Some genes involved in complex human diseases are particularly vulnerable to genetic variations such as single nucleotide polymorphism, copy number variations, and mutations. For example, Ras mutations account for over 30 % of all human cancers. Additionally, there are some genes that can display different variations with functional impact in different diseases that are unrelated. One such gene stands out: δ-catenin/NPRAP/Neurojungin with gene designation as CTNND2 on chromosome 5p15.2. Recent advances in genome wide association as well as molecular biology approaches have uncovered striking involvement of δ-catenin gene variations linked to complex human disorders. These disorders include cancer, bipolar disorder, schizophrenia, autism, Cri-du-chat syndrome, myopia, cortical cataract-linked Alzheimer's disease, and infectious diseases. This list has rapidly grown longer in recent years, underscoring the pivotal roles of δ-catenin in critical human diseases. δ-Catenin is an adhesive junction-associated protein in the delta subfamily of the β-catenin superfamily. δ-Catenin functions in Wnt signaling to regulate gene expression and modulate Rho GTPases of the Ras superfamily in cytoskeletal reorganization. δ-Catenin likely lies where Wnt signaling meets Rho GTPases and is a unique and vulnerable common target for mutagenesis in different human diseases. PMID:27380241

  18. Immunohistochemical expression of E-cadherin and β-catenin in feline mammary tumours.

    PubMed

    Zappulli, V; De Cecco, S; Trez, D; Caliari, D; Aresu, L; Castagnaro, M

    2012-01-01

    E-cadherin and β-catenin have been studied in carcinogenesis and tumour progression and reduced membrane expression of these molecules in canine mammary tumours has been associated with a poor prognosis. The present study investigated immunohistochemically the expression of E-cadherin and β-catenin in 53 mammary tumours and 48 hyperplastic or dysplastic lesions from 57 queens. E-cadherin and β-catenin expression was membranous in all samples and there was a significant decrease in expression in malignant tumours and metastases. Cytoplasmic expression of both markers was inversely correlated to the membrane localization. β-catenin nuclear labelling was detected in one lymph node metastasis (60% positive cells) and in the basal/myoepithelial cells of 6/7 ductal tumours. No correlation with survival was found for either marker. These results confirm the role of these proteins in maintaining tissue architecture and in inhibiting cell invasiveness and potentially indicate the oncogenic potential of the Wnt/β-catenin transduction pathway in feline mammary tumours. In addition, specific independent expression of β-catenin in the nuclei of basal/myoepithelial cells might suggest that this molecule is involved in regulation of the mammary stem/pluripotent cell component. Further studies should include more cases of benign mammary neoplasia and further investigate β-catenin nuclear expression in ductal tumours. PMID:22520821

  19. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  20. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones. PMID:26502645

  1. Transcriptional regulation of dentin matrix protein 1 (DMP1) in odontoblasts and osteoblasts.

    PubMed

    Jacob, Alexander; Zhang, Youbin; George, Anne

    2014-08-01

    Dentin matrix protein 1 (DMP1) is a noncollagenous protein important for the mineralization of bones and teeth. Examination of the transcription factor binding sites within the 6.24 kb upstream sequence of rat DMP1 promoter by Matinspector software revealed that TCF11 had the highest number (six) of binding sites with 100% matrix similarity. Four of these sites are conserved in the mouse DMP1 promoter. TCF11 is a member of the Cap-n-Collar (cnc) family of basic leucine zipper transcription factors. Results from this study showed that TCF11 can bind specifically to the DMP1 promoter and activate its transcription in odontoblasts and osteoblasts. This could be attributed to both direct and indirect effects of TCF11. Electrophoretic mobility shift (EMSA) assay showed differential interaction between TCF11 and its binding sites on the DMP1 promoter. 21 bp oligos spanning the TCF11 matrix were used as probes in EMSA, and the results showed that the binding was specific to the sequence of the TCF11 matrix as well as the flanking sequences and this is typical of a heterodimer binding site. Results also showed changes in the binding pattern when cells were differentiated in osteogenic medium for 2 d. Thus, TCF11 may play an important role in the transcriptional regulation of DMP1 gene.

  2. Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

    PubMed

    Getzenberg, R H; Coffey, D S

    1990-09-01

    The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

  3. Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy.

    PubMed

    Abeliovich, Hagai; Zarei, Mostafa; Rigbolt, Kristoffer T G; Youle, Richard J; Dengjel, Joern

    2013-01-01

    Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells, and defects in mitophagy correlate with ageing phenomena and with several neurodegenerative disorders. A central mechanistic question regarding mitophagy is whether mitochondria are consumed en masse, or whether an active process segregates defective molecules from functional ones within the mitochondrial network, thus allowing a more efficient culling mechanism. Here we combine a proteomic study with a molecular genetics and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We find that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism coupling weak physical segregation with mitochondrial dynamics to achieve a distillation-like effect. In agreement, the rates of mitophagic degradation strongly correlate with the degree of physical segregation of specific matrix proteins. PMID:24240771

  4. Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy

    NASA Astrophysics Data System (ADS)

    Abeliovich, Hagai; Zarei, Mostafa; Rigbolt, Kristoffer T. G.; Youle, Richard J.; Dengjel, Joern

    2013-11-01

    Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells, and defects in mitophagy correlate with ageing phenomena and with several neurodegenerative disorders. A central mechanistic question regarding mitophagy is whether mitochondria are consumed en masse, or whether an active process segregates defective molecules from functional ones within the mitochondrial network, thus allowing a more efficient culling mechanism. Here we combine a proteomic study with a molecular genetics and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We find that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism coupling weak physical segregation with mitochondrial dynamics to achieve a distillation-like effect. In agreement, the rates of mitophagic degradation strongly correlate with the degree of physical segregation of specific matrix proteins.

  5. High-throughput virtual screening and docking studies of matrix protein vp40 of ebola virus

    PubMed Central

    Tamilvanan, Thangaraju; Hopper, Waheeta

    2013-01-01

    Ebolavirus, a member of the Filoviridae family of negative-sense RNA viruses, causes severe haemorrhagic fever leading up to 90% lethality. Ebolavirus matrix protein VP40 is involved in the virus assembly and budding process. The RNA binding pocket of VP40 is considered as the drug target site for structure based drug design. High Throughput Virtual Screening and molecular docking studies were employed to find the suitable inhibitors against VP40. Ten compounds showing good glide score and glide energy as well as interaction with specific amino acid residues were short listed as drug leads. These small molecule inhibitors could be potent inhibitors for VP40 matrix protein by blocking virus assembly and budding process. PMID:23559747

  6. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  7. Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion.

    PubMed

    Clarke, Donald Nathaniel; Miller, Phillip W; Lowe, Christopher J; Weis, William I; Nelson, William James

    2016-08-01

    The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

  8. Characterization of the Cadherin–Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell–Cell Adhesion

    PubMed Central

    Clarke, Donald Nathaniel; Miller, Phillip W.; Lowe, Christopher J.; Weis, William I.; Nelson, William James

    2016-01-01

    The cadherin–catenin complex (CCC) mediates cell–cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis. We demonstrated that N. vectensis has a complete repertoire of cadherin–catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

  9. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    PubMed Central

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  10. Substratum Stiffness and Latrunculin B Regulate Matrix Gene and Protein Expression in Human Trabecular Meshwork Cells

    PubMed Central

    Thomasy, Sara M.; Wood, Joshua A.; Kass, Philip H.; Murphy, Christopher J.

    2012-01-01

    Purpose. To determine the impact of substratum stiffness and latrunculin-B (Lat-B), on the expression of several matrix proteins that are associated with glaucoma. Methods. Human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in dimethyl sulfoxide (DMSO) or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of secreted protein, acidic, cysteine rich (SPARC), myocilin, angiopoietin-like factor (ANGPTL)-7, and transglutaminase (TGM)-2. Immunofluorescence was used to assess changes in protein expression. Results. SPARC and myocilin mRNA expression were dramatically increased on the 75 kPa hydrogels and decreased on the 5 kPa hydrogels in comparison to TCP. In contrast, ANGPTL-7 mRNA and TGM-2 mRNA was decreased on the 75 kPa and 5 kPa hydrogels, respectively, in comparison with TCP. Treatment with Lat-B dramatically downregulated both SPARC and myocilin on 75 kPa hydrogels. In contrast, cells grown on TCP produced greater or similar amounts of SPARC and myocilin mRNA after Lat-B treatment. SPARC and myocilin protein expression paralleled changes in mRNA expression. Conclusions. Substratum stiffness impacts HTM matrix gene and protein expression and modulates the impact of Lat-B treatment on the expression of these matrix proteins. Integrating the use of biologically relevant substratum stiffness in the conduction of in vitro experiments gives important insights into HTM cell response to drugs that may more accurately predict responses observed in vivo. PMID:22247475

  11. Lack of anti-tumor activity with the β-catenin expression inhibitor EZN-3892 in the C57BL/6J Min/+ model of intestinal carcinogenesis

    SciTech Connect

    Hasson, Rian M.; Briggs, Alexandra; Rizvi, Hira; Carothers, Adelaide M.; Davids, Jennifer S.; Bertagnolli, Monica M.; Cho, Nancy L.

    2014-02-14

    Highlights: • Wnt/β-catenin signaling is aberrantly activated in most colorectal cancers. • Locked nucleic acid (LNA)-based antisense is a novel tool for cancer therapy. • β-Catenin inhibition was observed in mature intestinal tissue of LNA-treated mice. • Further investigation of Wnt/β-catenin targeted therapies is warranted. - Abstract: Background: Previously, we showed that short-term inhibition of β-catenin expression and reversal of aberrant β-catenin subcellular localization by the selective COX-2 inhibitor celecoxib is associated with adenoma regression in the C57BL/6J Min/+ mouse. Conversly, long-term administration resulted in tumor resistance, leading us to investigate alternative methods for selective β-catenin chemoprevention. In this study, we hypothesized that disruption of β-catenin expression by EZN-3892, a selective locked nucleic acid (LNA)-based β-catenin inhibitor, would counteract the tumorigenic effect of Apc loss in Min/+ adenomas while preserving normal intestinal function. Materials and methods: C57BL/6J Apc{sup +/+} wild-type (WT) and Min/+ mice were treated with the maximum tolerated dose (MTD) of EZN-3892 (30 mg/kg). Drug effect on tumor numbers, β-catenin protein expression, and nuclear β-catenin localization were determined. Results: Although the tumor phenotype and β-catenin nuclear localization in Min/+ mice did not change following drug administration, we observed a decrease in β-catenin expression levels in the mature intestinal tissue of treated Min/+ and WT mice, providing proof of principle regarding successful delivery of the LNA-based antisense vehicle. Higher doses of EZN-3892 resulted in fatal outcomes in Min/+ mice, likely due to β-catenin ablation in the intestinal tissue and loss of function. Conclusions: Our data support the critical role of Wnt/β-catenin signaling in maintaining intestinal homeostasis and highlight the challenges of effective drug delivery to target disease without permanent

  12. S-Adenosylmethionine and Methylthioadenosine Inhibit β-Catenin Signaling by Multiple Mechanisms in Liver and Colon Cancer

    PubMed Central

    Li, Tony W. H.; Peng, Hui; Yang, Heping; Kurniawidjaja, Steven; Panthaki, Parizad; Zheng, Yuhua; Mato, José M.

    2015-01-01

    S-Adenosylmethionine (SAMe), the principal methyl donor that is available as a nutritional supplement, and its metabolite methylthioadenosine (MTA) exert chemopreventive properties against liver and colon cancer in experimental models. Both agents reduced β-catenin expression on immunohistochemistry in a murine colitis-associated colon cancer model. In this study, we examined the molecular mechanisms involved. SAMe or MTA treatment in the colitis-associated cancer model lowered total β-catenin protein levels by 47 and 78%, respectively. In an orthotopic liver cancer model, increasing SAMe levels by overexpressing methionine adenosyltransferase 1A also reduced total β-catenin levels by 68%. In both cases, lower cyclin D1 and c-Myc expression correlated with lower β-catenin levels. In liver (HepG2) and colon (SW480, HCT116) cancer cells with constitutively active β-catenin signaling, SAMe and MTA treatment inhibited β-catenin activity by excluding it from the nuclear compartment. However, in liver (Huh-7) and colon (RKO) cancer cells expressing wild-type Wnt/β-catenin, SAMe and MTA accelerated β-catenin degradation by a glycogen synthase kinase 3-β–dependent mechanism. Both agents lowered protein kinase B activity, but this was not mediated by inhibiting phosphoinositide 3-kinase. Instead, both agents increased the activity of protein phosphatase 2A, which inactivates protein kinase B. The effect of MTA on lowering β-catenin is direct and not mediated by its conversion to SAMe, as blocking this conversion had no influence. In conclusion, SAMe and MTA inhibit Wnt/β-catenin signaling in colon and liver cancer cells regardless of whether this pathway is aberrantly induced, making them ideal candidates for chemoprevention and/or chemotherapy in these cancers. PMID:25338671

  13. Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

    PubMed Central

    Ren, Yizhong; Feng, Liqiang; Pan, Weiqi; Li, Liang; Wang, Qian; Li, Jiashun; Li, Na; Han, Ling; Zheng, Xuehua; Niu, Xuefeng; Sun, Caijun

    2015-01-01

    Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy. PMID:26468520

  14. Cell density modulates growth, extracellular matrix, and protein synthesis of cultured rat mesangial cells.

    PubMed

    Wolthuis, A; Boes, A; Grond, J

    1993-10-01

    Mesangial cell (MC) hyperplasia and accumulation of extracellular matrix are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of cell density on growth, extracellular matrix formation, and protein synthesis of cultured rat MCs. A negative linear relationship was found between initial plating density and DNA synthesis per cell after 24 hours incubation in medium with 10% fetal calf serum (range: 1 x 10(3) to 7 x 10(5) MCs/2cm2, r = 0.996, P < 0.001). Enzyme-linked immunosorbent assay of the amount of fibronectin in the conditioned medium after 72 hours showed a negative relationship with increasing cell density. In contrast, the amount of cell-associated fibronectin increased to maximal values in confluent cultures, and no further increase was seen at supraconfluency. The relative collagen synthesis in the conditioned medium and cell layer--assessed by collagenase digestion after 5 hours [3H]proline pulse labeling--showed a similar pattern. Secreted collagen decreased with increasing cell density from 3.4% to 0.2% of total protein synthesis. In contrast, cell-associated collagen increased from 1.1% to 11.8% of newly synthesized protein until confluency followed by a decrease to 4.2% at supraconfluency. Specific immunoprecipitation of collagen types I, III, and IV revealed a significant (twofold) increase in collagen I synthesis per cell at confluency. Collagen III and IV synthesis was not affected by cell density. Specific protein expression in both the medium and cell layer were analyzed by two-dimensional polyacrylamide gel electrophoresis (150 to 20 kd, pI 5.0 to 7.0) after 20 hours steady-state metabolic labeling with [35S]methionine. Supraconfluent MCs displayed overexpression of 10, underexpression of four, new expression of five, and changed mobility of three different intracellular proteins. Of interest was the overexpression of two proteins (89 kd, pI 5.31 and 72 kd, pI 5.32) that were identified by immunoblotting as

  15. A Cell-Free Assay System for β-Catenin Signaling That Recapitulates Direct Inductive Events in the Early Xenopus laevis Embryo

    PubMed Central

    Nelson, Richard W.; Gumbiner, Barry M.

    1999-01-01

    In vertebrate embryos, signaling via the β-catenin protein is known to play an essential role in the induction of the dorsal axis. In its signaling capacity, β-catenin acts directly to affect target gene transcription, in concert with transcription factors of the TCF/LEF family. We have developed a cell-free in vitro assay for β-catenin signaling activity that utilizes transcriptionally active nuclei and cytoplasm from cleavage-blocked Xenopus laevis embryos. Under these assay conditions, we demonstrate that either addition of β-catenin protein or upstream activation of the β-catenin signaling pathway can induce the expression of developmentally relevant target genes. Addition of exogenous β-catenin protein induced expression of Siamois, XTwin, Xnr3, and Cerberus mRNAs in a protein synthesis independent manner, whereas a panel of other Spemann organizer-specific genes did not respond to β-catenin. Lithium induction of the β-catenin signaling pathway, which is thought to cause β-catenin accumulation by inhibiting its proteasome-dependent degradation, caused increased expression of Siamois in a protein synthesis independent fashion. This result suggests that β-catenin derived from a preexisting pool can be activated to signal, and that accumulation of this activated form does not require ongoing synthesis. Furthermore, activation of the signaling pathway with lithium did not detectably alter cytoplasmic β-catenin levels and was insensitive to inhibition of the proteasome- dependent degradation pathway. Taken together, these results suggest that activation of β-catenin signaling by lithium in this system may occur through a distinct activation mechanism that does not require modulation of levels through regulation of proteasomal degradation. PMID:10525541

  16. Loss of αT-catenin alters the hybrid adhering junctions in the heart and leads to dilated cardiomyopathy and ventricular arrhythmia following acute ischemia.

    PubMed

    Li, Jifen; Goossens, Steven; van Hengel, Jolanda; Gao, Erhe; Cheng, Lan; Tyberghein, Koen; Shang, Xiying; De Rycke, Riet; van Roy, Frans; Radice, Glenn L

    2012-02-15

    It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The α-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.

  17. FOXP1 potentiates Wnt/β-catenin signaling in diffuse large B cell lymphoma.

    PubMed

    Walker, Matthew P; Stopford, Charles M; Cederlund, Maria; Fang, Fang; Jahn, Christopher; Rabinowitz, Alex D; Goldfarb, Dennis; Graham, David M; Yan, Feng; Deal, Allison M; Fedoriw, Yuri; Richards, Kristy L; Davis, Ian J; Weidinger, Gilbert; Damania, Blossom; Major, Michael B

    2015-02-03

    The transcription factor FOXP1 (forkhead box protein P1) is a master regulator of stem and progenitor cell biology. In diffuse large B cell lymphoma (DLBCL), copy number amplifications and chromosomal translocations result in overexpression of FOXP1. Increased abundance of FOXP1 in DLBCL is a predictor of poor prognosis and resistance to therapy. We developed a genome-wide, mass spectrometry-coupled, gain-of-function genetic screen, which revealed that FOXP1 potentiates β-catenin-dependent, Wnt-dependent gene expression. Gain- and loss-of-function studies in cell models and zebrafish confirmed that FOXP1 was a general and conserved enhancer of Wnt signaling. In a Wnt-dependent fashion, FOXP1 formed a complex with β-catenin, TCF7L2 (transcription factor 7-like 2), and the acetyltransferase CBP [CREB (adenosine 3',5'-monophosphate response element-binding protein)-binding protein], and this complex bound the promoters of Wnt target genes. FOXP1 promoted the acetylation of β-catenin by CBP, and acetylation was required for FOXP1-mediated potentiation of β-catenin-dependent transcription. In DLBCL, we found that FOXP1 promoted sensitivity to Wnt pathway inhibitors, and knockdown of FOXP1 or blocking β-catenin transcriptional activity slowed xenograft tumor growth. These data connect excessive FOXP1 with β-catenin-dependent signal transduction and provide a molecular rationale for Wnt-directed therapy in DLBCL.

  18. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair.

    PubMed

    Caves, Jeffrey M; Cui, Wanxing; Wen, Jing; Kumar, Vivek A; Haller, Carolyn A; Chaikof, Elliot L

    2011-08-01

    Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23-314%), six-fold in Young's modulus (5.3-33.1 MPa), and more than two-fold in tensile strength (1.85-4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity. PMID:21550111

  19. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair

    PubMed Central

    Caves, Jeffrey M.; Cui, Wanxing; Wen, Jing; Kumar, Vivek A.; Haller, Carolyn A.; Chaikof, Elliot L.

    2011-01-01

    Artificial composites designed to mimic the structure and properties of native extracellular matrix may lead to acellular materials for soft tissue repair and replacement, which display mechanical strength, stiffness, and resilience resembling native tissue. We describe the fabrication of thin lamellae consisting of continuous collagen microfiber embedded at controlled orientations and densities in a recombinant elastin-like protein polymer matrix. Multilamellar stacking affords flexible, protein-based composite sheets whose properties are dependent upon both the elastomeric matrix and collagen content and organization. Sheets are produced with properties that range over 13-fold in elongation to break (23 – 314%), six-fold in Young’s modulus (5.3 to 33.1 MPa), and more than two-fold in tensile strength (1.85 to 4.08 MPa), exceeding that of a number of native human tissues, including urinary bladder, pulmonary artery, and aorta. A sheet approximating the mechanical response of human abdominal wall fascia is investigated as a fascial substitute for ventral hernia repair. Protein-based composite patches prevent hernia recurrence in Wistar rats over an 8-week period with new tissue formation and sustained structural integrity. PMID:21550111

  20. Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins.

    PubMed

    Seong, Jihye; Tajik, Arash; Sun, Jie; Guan, Jun-Lin; Humphries, Martin J; Craig, Susan E; Shekaran, Asha; García, Andrés J; Lu, Shaoying; Lin, Michael Z; Wang, Ning; Wang, Yingxiao

    2013-11-26

    Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.

  1. Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1

    NASA Astrophysics Data System (ADS)

    He, Gen; Dahl, Tom; Veis, Arthur; George, Anne

    2003-08-01

    Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Acidic extracellular matrix proteins play a pivotal role during biomineral formation. However, the mechanisms of protein-mediated mineral initiation are far from understood. Here we report that dentin matrix protein 1 (DMP1), an acidic protein, can nucleate the formation of hydroxyapatite in vitro in a multistep process that begins by DMP1 binding calcium ions and initiating mineral deposition. The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Subsequently, these expand and coalesce into microscale crystals elongated in the c-axis direction. Characterization of the functional domains in DMP1 demonstrated that intermolecular assembly of acidic clusters into a β-sheet template was essential for the observed mineral nucleation. Protein-mediated initiation of nanocrystals, as discussed here, might provide a new methodology for constructing nanoscale composites by self-assembly of polypeptides with tailor-made peptide sequences.

  2. MVsCarta: A protein database of matrix vesicles to aid understanding of biomineralization.

    PubMed

    Cui, Yazhou; Xu, Quan; Luan, Jing; Hu, Shichang; Pan, Jianbo; Han, Jinxiang; Ji, Zhiliang

    2015-06-01

    Matrix vesicles (MVs) are membranous nanovesicles released by chondrocytes, osteoblasts, and odontoblasts. They play a critical role in modulating mineralization. Here, we present a manually curated database of MV proteins, namely MVsCara to provide comprehensive information on MVs of protein components. In the current version, the database contains 2,713 proteins of six organisms identified in bone, cartilage, tooth tissues, and cells capable of producing a mineralized bone matrix. The MVsCarta database is now freely assessed at http://bioinf.xmu.edu.cn/MVsCarta. The search and browse methods were developed for better retrieval of data. In addition, bioinformatic tools like Gene Ontology (GO) analysis, network visualization and protein-protein interaction analysis were implemented for a functional understanding of MVs components. Similar database hasn't been reported yet. We believe that this free web-based database might serve as a useful repository to elucidate the novel function and regulation of MVs during mineralization, and to stimulate the advancement of MV studies. PMID:26166372

  3. Trends in global warming and evolution of matrix protein 2 family from influenza A virus.

    PubMed

    Yan, Shao-Min; Wu, Guang

    2009-12-01

    The global warming is an important factor affecting the biological evolution, and the influenza is an important disease that threatens humans with possible epidemics or pandemics. In this study, we attempted to analyze the trends in global warming and evolution of matrix protein 2 family from influenza A virus, because this protein is a target of anti-flu drug, and its mutation would have significant effect on the resistance to anti-flu drugs. The evolution of matrix protein 2 of influenza A virus from 1959 to 2008 was defined using the unpredictable portion of amino-acid pair predictability. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site, and species carrying influenza A virus. The results showed the similar trends in global warming and in evolution of M2 proteins although we could not correlate them at this stage of study. The study suggested the potential impact of global warming on the evolution of proteins from influenza A virus.

  4. Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum*

    PubMed Central

    Debreuil, Julien; Tambutté, Éric; Zoccola, Didier; Deleury, Emeline; Guigonis, Jean-Marie; Samson, Michel; Allemand, Denis; Tambutté, Sylvie

    2012-01-01

    We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum. PMID:22505718

  5. Prenylated Rab acceptor 1 (PRA1) inhibits TCF/{beta}-catenin signaling by binding to {beta}-catenin

    SciTech Connect

    Kim, Jong-Tae; Cho, Mi-Young; Choi, Seung-Chul; Kim, Jung Woo; Chae, Suhn-Kee; Yoon, Do-Young; Kim, Jae Wha . E-mail: wjkim@kribb.re.kr; Lim, Jong-Seok . E-mail: jslim@sookmyung.ac.kr

    2006-10-13

    The prenylated Rab acceptor 1 (PRA1) is a ubiquitously expressed 21 kDa protein containing two transmembrane domains that possibly induce its localization to the Golgi complex. It binds to prenylated Rab GTPases and VAMP2. In this study, we report that PRA1-overexpressing cells exhibited a significantly retarded growth rate as compared to that of the mock-transfected cells, and the transcriptional activity of TCF, as evaluated by TOPflash luciferase reporter assay, was profoundly reduced in the PRA1-overexpressed cells. These intracellular functions of PRA1 were verified by introducing deletion mutant or site-directed mutants, or small interfering RNA of PRA1. In addition, the translocation of {beta}-catenin from the cytosol to the nucleus was blocked to a significant degree in the PRA1-cells, and the interaction of PRA1 and {beta}-catenin was identified by confocal microscopy and immunoprecipitation analysis. Finally, we observed that the inhibition of TCF/{beta}-catenin signaling by PRA1 is associated with ERK1/2 dephosphorylation. Therefore, our data suggest that the in vivo modulation of PRA1 may be involved in TCF/{beta}-catenin signaling, as well as cellular proliferation and tumorigenesis.

  6. Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study.

    PubMed

    Haldar, Sourav; Chattopadhyay, Amitabha

    2007-12-27

    The fluorophore in green fluorescent protein (GFP) is localized in a highly constrained environment, protected from the bulk solvent by the barrel-shaped protein matrix. We have used the wavelength-selective fluorescence approach (red edge excitation shift, REES) to monitor solvent (environment) dynamics around the fluorophore in enhanced green fluorescent protein (EGFP) under various conditions. Our results show that EGFP displays REES in buffer and glycerol, i.e., the fluorescence emission maxima exhibit a progressive shift toward the red edge, as the excitation wavelength is shifted toward the red edge of the absorption spectrum. Interestingly, EGFP displays REES when incorporated in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT), independent of the hydration state. We interpret the observed REES to the constrained environment experienced by the EGFP fluorophore in the rigid protein matrix, rather than to the dynamics of the bulk solvent. These results are supported by the temperature dependence of REES and characteristic wavelength-dependent changes in fluorescence anisotropy.

  7. Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

    PubMed Central

    Miyagawa, S; Harada, M; Matsumaru, D; Tanaka, K; Inoue, C; Nakahara, C; Haraguchi, R; Matsushita, S; Suzuki, K; Nakagata, N; Ng, R C-L; Akita, K; Lui, V C-H; Yamada, G

    2014-01-01

    The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (ShhCreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The ShhCreERT2/+; β-cateninflox(ex3)/+; BmprIAflox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to

  8. Identification of the regions of PECAM-1 involved in beta- and gamma-catenin associations.

    PubMed

    Biswas, Purba; Zhang, Jin; Schoenfeld, Jonathan D; Schoenfeld, David; Gratzinger, Dita; Canosa, Sandra; Madri, Joseph A

    2005-04-22

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated beta-catenin and modulates beta-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated beta-catenin, and to a lesser extent unphosphorylated beta-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for beta-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, gamma-catenin-PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1-gamma-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S(673) to C and D and S(669 and 670) to C, we confirmed the importance of S(673) and its phosphorylation state as a mediator of gamma-catenin-PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1-catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions. PMID:15766557

  9. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  10. Parathyroid hormone regulates the expression of rat osteoblast and osteosarcoma nuclear matrix proteins.

    PubMed

    Bidwell, J; Feister, H; Swartz, D; Onyia, J; Holden, J; Hock, J

    1996-12-01

    Parathyroid hormone (PTH) alters osteoblast morphology. How these changes in cell shape modify nuclear structure and ultimately gene expression is not known. Chronic exposure to rat PTH (1-34) [10 nM] attenuated the expression of 200, 190, and 160 kD proteins in the nuclear matrix-intermediate filament subfraction of the rat osteosarcoma cells, ROS 17/2.8 [Bidwell et al. (1994b): Endocrinology 134:1738-1744]. Here, we determined that these same PTH-responsive proteins were expressed in rat metaphyseal osteoblasts. We identified the 200 kD protein as a non-muscle myosin. Although the molecular weights, subcellular distribution, and half-lives of the 190 and 160 kD proteins were similar to topoisomerase II-alpha and -beta, nuclear matrix enzymes that mediate DNA topology, the 190 and 160 kD proteins did not interact with topoisomerase antibodies. Nevertheless, the expression of topoisomerase II-alpha, and NuMA, a component of the nuclear core filaments, was also regulated by PTH in the osteosarcoma cells. The 190 kD protein was selectively expressed in bone cells as it was not observed in OK opossum kidney cells, H4 hepatoma cells, or NIH3T3 cells. PTH attenuated mRNA expression of the PTH receptor in our cell preparations. These results demonstrate that PTH selectively alters the expression of osteoblast membrane, cytoskeletal, and nucleoskeletal proteins. Topoisomerase II-alpha, NuMA, and the 190 and 160 kD proteins may direct the nuclear PTH signalling pathways to the target genes and play a structural role in osteoblast gene expression. PMID:8913889

  11. Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis.

    PubMed

    Komarova, Anastassia V; Real, Eléonore; Borman, Andrew M; Brocard, Michèle; England, Patrick; Tordo, Noël; Hershey, John W B; Kean, Katherine M; Jacob, Yves

    2007-01-01

    Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.

  12. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  13. 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.

  14. Modulation of matrix mineralization by Vwc2-like protein and its novel splicing isoforms

    PubMed Central

    Ohyama, Yoshio; Katafuchi, Michitsuna; Almehmadi, Ahmed; Venkitapathi, Sundharamani; Jaha, Haytham; Ehrenman, Jason; Morcos, Joseph; Aljamaan, Reem; Mochida, Yoshiyuki

    2012-01-01

    In search of new cysteine knot protein (CKP) family members, we found a novel gene called von Willebrand factor C domain-containing protein 2-like (Vwc2l, also known as Brorin-like) and its transcript variants (Vwc2l-1, Vwc2l-2 and Vwc2l-3). Based on the deduced amino acid sequence, Vwc2l-1 has a signal peptide and 2 cysteine-rich (CR) domains, while Vwc2l-2 lacks a part of 2nd CR domain and Vwc2l-3 both CR domains. Although it has been reported that the expression of Brorin-like was predominantly observed in brain, we found that Vwc2l transcript variants were detected in more ubiquitous tissues. In osteoblasts, the induction of Vwc2l expression was observed at matrix mineralization stage. When Vwc2l was stably transfected into osteoblasts, the matrix mineralization was markedly accelerated in Vwc2l-expressing clones compared to that in the control, indicating the modulatory effect of Vwc2l protein on osteoblastic cell function. The mechanistic insight of Vwc2l-modulation was further investigated and we found that the expression of Osterix, one of the key osteogenic markers, was significantly increased by addition of all Vwc2l isoform proteins. Taken together, Vwc2l is a novel secreted protein that promotes matrix mineralization by modulating Osterix expression likely through TGF-β superfamily growth factor signaling pathway. Our data may provide mechanistic insights into the biological functions of this novel CKP member in bone and further suggest a novel approach to enhance osteoblast function, which enables to accerelate bone formation, regeneration and healing. PMID:22209847

  15. Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

    PubMed Central

    Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

    2014-01-01

    ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998

  16. C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

    PubMed Central

    Ray, Greeshma; Schmitt, Phuong Tieu

    2016-01-01

    ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein

  17. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein

    PubMed Central

    2013-01-01

    Background Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Results Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. Conclusion The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic

  18. 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

  19. Constitutive Nuclear Expression of Dentin Matrix Protein 1 Fails to Rescue the Dmp1-null Phenotype*

    PubMed Central

    Lin, Shuxian; Zhang, Qi; Cao, Zhengguo; Lu, Yongbo; Zhang, Hua; Yan, Kevin; Liu, Ying; McKee, Marc D.; Qin, Chunlin; Chen, Zhi; Feng, Jian Q.

    2014-01-01

    Dentin matrix protein 1 (DMP1) plays multiple roles in bone, tooth, phosphate homeostasis, kidney, salivary gland, reproductive cycles, and the development of cancer. In vitro studies have indicated two different biological mechanisms: 1) as a matrix protein, DMP1 interacts with αvβ3 integrin and activates MAP kinase signaling; and 2) DMP1 serves as a transcription co-factor. In vivo studies have demonstrated its key role in osteocytes. This study attempted to determine whether DMP1 functions as a transcription co-factor and regulates osteoblast functions. For gene expression comparisons using adenovirus constructs, we targeted the expression of DMP1 either to the nucleus only by replacing the endogenous signal peptide with a nuclear localization signal (NLS) sequence (referred to as NLSDMP1) or to the extracellular matrix as the WT type (referred to as SPDMP1) in MC3T3 osteoblasts. High levels of DMP1 in either form greatly increased osteogenic gene expression in an identical manner. However, the targeted NLSDMP1 transgene driven by a 3.6-kb rat Col 1α1 promoter in the nucleus of osteoblasts and osteocytes failed to rescue the phenotyope of Dmp1-null mice, whereas the SPDMP1 transgene rescued the rickets defect. These studies support the notion that DMP1 functions as an extracellular matrix protein, rather than as a transcription co-factor in vivo. We also show that DMP1 continues its expression in osteoblasts during postnatal development and that the deletion of Dmp1 leads to an increase in osteoblast proliferation. However, poor mineralization in the metaphysis indicates a critical role for DMP1 in both osteoblasts and osteocytes. PMID:24917674

  20. The positive correlation between DJ-1 and β-catenin expression shows prognostic value for patients with glioma.

    PubMed

    Wang, Chao; Fang, Mao; Zhang, Meng; Li, Weiping; Guan, Hong; Sun, Yanhua; Xie, Siming; Zhong, Xueyun

    2013-12-01

    The relationship between DJ-1 and β-catenin, and its impact on the prognosis for glioma patients has not been fully understood. This study determined the effect of DJ-1 on β-catenin and the prognostic significance of this interaction in glioma patients. We collected tumor specimens from 88 glioma patients and determined the expression of DJ-1, β-catenin and PTEN by using immunohistochemical staining. The involvement of DJ-1 and β-catenin in glioma cell lines was evaluated by immunohistochemistry and Western blotting. High DJ-1 expression (37.5%) and high β-catenin expression (34.1%) in glioma specimens were significantly associated with high grade and poor prognosis in glioma patients. However, only high levels of DJ-1 (P = 0.014) was a strong independent prognostic factor, correlated with a reduced overall survival time. In vitro DJ-1 expression was positively correlated with the expression levels of β-catenin and p-Akt, and negatively correlated with PTEN expression in U87, U251 MG, SWO-38 and SHG44 human glioma cell lines. After the knockdown of DJ-1, Akt, p-Akt or β-catenin expression levels were not affected in the PTEN-null cell lines (U87 and U251 MG). However, in the SWO-38 cell line, which has wild-type PTEN protein, the level of PTEN increased while Akt/p-Akt and β-catenin levels were reduced. Furthermore, β-catenin staining weakened in SWO-38 cells after DJ-1 levels decreased according to immunocytochemical analysis. In conclusion, DJ-1 and β-catenin may contribute to the development and recurrence of glioma and are valuable prognostic factors for glioma patients. DJ-1 may regulate β-catenin expression via PTEN and p-Akt.

  1. 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.

  2. Analysis of matrix proteins of otolith in upside-down catfish

    NASA Astrophysics Data System (ADS)

    Ohnishi, K.; Okamoto, N.; Takahashi, A.; Ohnishi, T.

    We have previously suggested that the calcium density of the otolith in upside-down swimming Synodontis nigriventris is lower than that in upside-up swimming Synodontis multipunctatus Biol Space Sci 2002 In this study we examined EDTA-soluble matrix proteins of otolith in the utricle of the catfish S nigriventris S multipunctatus and upside-up swimming Synodontis brichadi and goldfish Carassius auratus We detected two main bands about 55 kD and 80 kD with SDS-PAGE in the 3 species of the catfish In cntrast goldfish had the about 55 kD band alone The band of about 80 kD was consisted of two sub-bands a lighter and a heavier band A lighter band was observed in S brichadi and a heavier band was observed in S nigriventris S multipunctatus had the both bands Furthermore mass spectrometric analysis showed there were some proteins of molecular weight under 14 kD The molecular weights of the proteins were different among the fishes These results suggest that many different kinds of matrix protein may cause different degree of calcification in otolith formation

  3. LMAN1 (ERGIC-53) is a potential carrier protein for matrix metalloproteinase-9 glycoprotein secretion

    PubMed Central

    Duellman, Tyler; Burnett, John; Shin, Alice; Yang, Jay

    2015-01-01

    Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extra-cellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion. PMID:26150355

  4. Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins

    PubMed Central

    Shibata, Toshio; Maki, Kouki; Hadano, Jinki; Fujikawa, Takumi; Kitazaki, Kazuki; Koshiba, Takumi; Kawabata, Shun-ichiro

    2015-01-01

    Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes. PMID:26506243

  5. Protein-transitions in and out of the dough matrix in wheat flour mixing.

    PubMed

    Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

    2017-02-15

    Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour. PMID:27664670

  6. Protein-transitions in and out of the dough matrix in wheat flour mixing.

    PubMed

    Wang, Xiaolong; Appels, Rudi; Zhang, Xiaoke; Bekes, Ferenc; Torok, Kitti; Tomoskozi, Sandor; Diepeveen, Dean; Ma, Wujun; Islam, Shahidul

    2017-02-15

    Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour.

  7. Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases.

    PubMed

    Wille, Christoph; Köhler, Conny; Armacki, Milena; Jamali, Arsia; Gössele, Ulrike; Pfizenmaier, Klaus; Seufferlein, Thomas; Eiseler, Tim

    2014-02-01

    Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix-bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies. PMID:24336522

  8. Herpes simplex (HSV-1) infection of bovine aorta smooth muscle cells (SMC) inhibits matrix protein synthesis

    SciTech Connect

    Lashgari, M.S.; Friedman, H.M.; Kefalides, N.A.

    1986-03-01

    Studies from this laboratory have shown that HSV-1 infection suppresses matrix protein synthesis by endothelial cells in vitro. In this study the authors have investigated the effects of HSV-1 infection on SMC. Monolayers of SMC were infected with HSV-1 at a multiplicity of infection (MOI) ranging from 0.1 to 20. Viral replication and release to the medium was measured by plaque assay in Vero cells. At an MOI of 0.1, 10 or 20, viral replication occurred and maximum virus titers were achieved by 24 hrs. post-infection. Virus release in the medium began during the first 12 hrs. post-infection and reached maximum at 24 hrs. Infected and uninfected cultures of SMC were pulse labeled with either (/sup 14/C)proline or (/sup 35/S)-methionine at different hrs. post-infection. Incorporation of radioactivity into non-dialyzable protein was determined in fluorograms following SDS-PAGE of the cell-matrix or medium fractions. The synthesis of fibronectin and collagen Types I and III was suppressed and the degree of suppression was dependent on the duration of infection and on the virus dose. These data suggest that SMC can support HSV-1 replication in vitro and that such infection can lead to altered extracellular matrix synthesis.

  9. Retinoic Acid Ameliorates Pancreatic Fibrosis and Inhibits the Activation of Pancreatic Stellate Cells in Mice with Experimental Chronic Pancreatitis via Suppressing the Wnt/β-Catenin Signaling Pathway

    PubMed Central

    Yin, Guojian; Fan, Yuting; Wu, Deqing; Qiu, Lei; Yu, Ge; Xing, Miao; Hu, Guoyong; Wang, Xingpeng; Wan, Rong

    2015-01-01

    Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of β-catenin, platelet-derived growth factor (PDGF)-Rβ transforming growth factor (TGF)-βRII and collagen 1α1 in vivo. Wnt 2 and β-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of β-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFβRII, PDGFRβ and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/β-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice. PMID:26556479

  10. Retinoic Acid Ameliorates Pancreatic Fibrosis and Inhibits the Activation of Pancreatic Stellate Cells in Mice with Experimental Chronic Pancreatitis via Suppressing the Wnt/β-Catenin Signaling Pathway.

    PubMed

    Xiao, Wenqin; Jiang, Weiliang; Shen, Jie; Yin, Guojian; Fan, Yuting; Wu, Deqing; Qiu, Lei; Yu, Ge; Xing, Miao; Hu, Guoyong; Wang, Xingpeng; Wan, Rong

    2015-01-01

    Pancreatic fibrosis, a prominent feature of chronic pancreatitis (CP), induces persistent and permanent damage in the pancreas. Pancreatic stellate cells (PSCs) provide a major source of extracellular matrix (ECM) deposition during pancreatic injury, and persistent activation of PSCs plays a vital role in the progression of pancreatic fibrosis. Retinoic acid (RA), a retinoid, has a broad range of biological functions, including regulation of cell differentiation and proliferation, attenuating progressive fibrosis of multiple organs. In the present study, we investigated the effects of RA on fibrosis in experimental CP and cultured PSCs. CP was induced in mice by repetitive cerulein injection in vivo, and mouse PSCs were isolated and activated in vitro. Suppression of pancreatic fibrosis upon administration of RA was confirmed based on reduction of histological damage, α-smooth muscle actin (α-SMA) expression and mRNA levels of β-catenin, platelet-derived growth factor (PDGF)-Rβ transforming growth factor (TGF)-βRII and collagen 1α1 in vivo. Wnt 2 and β-catenin protein levels were markedly down-regulated, while Axin 2 expression level was up-regulated in the presence of RA, both in vivo and in vitro. Nuclear translation of β-catenin was significantly decreased following RA treatment, compared with cerulein-induced CP in mice and activated PSCs. Furthermore, RA induced significant PSC apoptosis, inhibited proliferation, suppressed TCF/LEF-dependent transcriptional activity and ECM production of PSC via down-regulation of TGFβRII, PDGFRβ and collagen 1α1 in vitro. These results indicate a critical role of the Wnt/β-catenin signaling pathway in RA-induced effects on CP and PSC regulation and support the potential of RA as a suppressor of pancreatic fibrosis in mice. PMID:26556479

  11. Chondroprotective effects of palmatine on osteoarthritis in vivo and in vitro: A possible mechanism of inhibiting the Wnt/β-catenin and Hedgehog signaling pathways.

    PubMed

    Zhou, Xindie; Lin, Xiaolong; Xiong, Yan; Jiang, Lifeng; Li, Weijun; Li, Jin; Wu, Lidong

    2016-05-01

    The present study aimed to investigate the effect of palmatine (Pal) in a rabbit osteoarthritis (OA) model in vivo and rabbit interleukin-1β (IL-1β)-stimulated chondrocytes in vitro. Appropriate concentrations of Pal were identified by the MTT assay and used to preincubate IL-1β-induced chondrocytes, as well as an activator or inhibitor of Wnt and Hedgehog signaling pathways. Matrix metalloproteinase (MMP)-1, 3, and 13; tissue inhibitor of metalloproteinase (TIMP)-1; collagenase II; aggrecan; and the related molecules of the Wnt/β-catenin and Hedgehog signaling pathways were investigated. Protein expression was detected by Western blot analysis and messenger RNA (mRNA) expression was examined by PCR analysis. Pal (0.3 mL, 100 mg/L) was injected into rabbit knee joints and histological examination, immunohistochemistry, and Mankin scoring of the articular cartilage were performed. Pal (10-100 mg/L) had no effect on chondrocyte viability, decreased the expression of the MMPs, and increased the synthesis of TIMP-1whereas collagenase II and aggrecan were inhibited by IL-1β. When the activator (Licl) and inhibitor (DKK-1) of the Wnt/β-catenin signaling pathway as well as the inhibitor (cyclopamine) of the Hedgehog signaling pathway were added, the Wnt/β-catenin signaling pathway was less inhibited by Pal, and a similar inhibitory effect of cyclopamine on the Hedgehog signaling pathway was evident. Additionally, Pal enhanced the effect of cyclopamine. The histological examination, immunohistochemistry and Mankin scoring also demonstrated the protective effect of Pal, and the inhibition of the Wnt and Hedgehog signaling pathways by Pal. Pal may be useful in the treatment of OA, in which its effect is likely mediated via the Wnt/β-catenin and Hedgehog signaling pathways. PMID:26945831

  12. Expression profiling of wild type and β-catenin gene disrupted human BxPC-3 pancreatic adenocarcinoma cells.

    PubMed

    Olsen, Petter Angell; Lund, Kaja; Krauss, Stefan

    2015-06-01

    To study the role of WNT/β-catenin signaling in pancreatic adenocarcinoma, human BxPC-3 cell lines deficient of the central canonical WNT signaling protein β-catenin were established by using zinc-finger nuclease mediated targeted genomic disruption of the β-catenin gene (CTNNB1). Comparison of the global transcription levels in wild type cells with two β-catenin gene disrupted clones identified 85 transcripts that were the most differentially regulated. Gene ontology (GO) term enrichment analysis of these transcripts identified "cell adhesion" as the most significantly enriched GO term. Here we describe the data from the transcription profiling analysis published in the article "Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells" [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE63072. PMID:26484203

  13. Genetic analysis of phosphoprotein and matrix protein of rabies viruses isolated in Brazil.

    PubMed

    Kobayashi, Yuki; Okuda, Hiromi; Nakamura, Kana; Sato, Go; Itou, Takuya; Carvalho, Adolorata A B; Silva, Marlon V; Mota, Carla S; Ito, Fumio H; Sakai, Takeo

    2007-11-01

    To investigate the genetic characteristics of phosphoprotein (P) and matrix protein (M) genes of variable rabies virus (RV) prevalent in Brazil, the authors genetically characterized the P and M genes from 30 Brazilian RV field isolates. Phylogenetic analysis based on the P and M genes revealed the presence of six RV variants that consisted primarily of three insectivorous bats, the vampire bat, dog and fox in Brazil. Specific amino acid substitutions corresponding to these phylogenetic lineages were observed, with Asp(42) and Glu(62) in the P protein found to be characteristic of Brazilian chiroptera- and carnivora-related RVs, respectively. Amino acid sequence motifs predicted to associate with a viral function in the P and M proteins were conserved among Brazilian RV variants. PMID:18057829

  14. Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

    PubMed Central

    2014-01-01

    Background Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data. Results A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport. Conclusions This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling. PMID:24712863

  15. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    NASA Astrophysics Data System (ADS)

    Nakanishi, Jun; Nakayama, Hidekazu; Yamaguchi, Kazuo; Garcia, Andres J.; Horiike, Yasuhiro

    2011-08-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG7). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7-10) to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  16. Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of β-catenin activity

    PubMed Central

    Johnson, Abby L.; Zinser, Glendon M.; Waltz, Susan E.

    2015-01-01

    The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct β-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and β-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes β-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active β-catenin were markedly increased in the absence of VDR. In vitro, VDR activation in breast cancer cells reduced β-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of β-catenin with the cyclin D1 promoter. Expression of a stabilized form or β-catenin ablated the protective effects of VDR activation. Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of β-catenin activation. PMID:26008979

  17. Effects of Bone Matrix Proteins on Fracture and Fragility in Osteoporosis

    PubMed Central

    Sroga, Grażyna E.

    2012-01-01

    Bone mineral density alone cannot reliably predict fracture risk in humans and laboratory animals. Therefore, other factors including the quality of organic bone matrix components and their interactions may be of crucial importance to understanding of fragility fractures. Emerging research evidence shows, that in addition to collagen, certain noncollagenous proteins (NCPs) play a significant role in the structural organization of bone and influence its mechanical properties. However, their contribution to bone strength still remains largely undefined. Collagen and NCPs undergo different post-translational modifications, which alter the quality of the extracellular matrix and the response of bone to mechanical load. The primary focus of this overview is on NCPs that, together with collagen, contribute to structural and mechanical properties of bone. Current information on several mechanisms through which some NCPs influence bone’s resistance to fracture, including the role of nonenzymatic glycation, is also presented. PMID:22535528

  18. Alterations of the Wnt/beta-catenin pathway and its target genes for the N- and C-terminal domains of parathyroid hormone-related protein in bone from diabetic mice.

    PubMed

    Portal-Núñez, S; Lozano, D; de Castro, L Fernández; de Gortázar, A R; Nogués, X; Esbrit, P

    2010-07-16

    Type 1 diabetes mellitus (T1D) is associated with bone loss. Given that the Wnt/beta-catenin pathway is a major regulator of bone accrual, we assessed this pathway in mice with streptozotozin-induced T1D. In diabetic mouse long bones, we found alterations favouring the suppression of this pathway by using PCR arrays and beta-catenin immunostaining. Downregulation of sclerostin, an inhibitor of this pathway, also occurred, and related to increased osteocyte apoptosis. Our data show that both N- and C-terminal parathyroid hormone-related peptide fragments might exert osteogenic effects in this setting by targeting several genes of this pathway and increasing beta-catenin in osteoblastic cells.

  19. S-nitrosation of β-catenin and p120 catenin: a novel regulatory mechanism in endothelial hyperpermeability

    PubMed Central

    Marín, N.; Zamorano, P.; Carrasco, R.; Mujica, P.; González, FG.; Quezada, C.; Meininger, CJ.; Boric, MP.; Durán, WN.; Sánchez, FA.

    2014-01-01

    Rationale Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of eNOS to cytosol and stimulation of eNOS-derived NO signaling cascade. The mechanisms by which NO signaling regulates permeability at adherens junctions are still incompletely understood. Objective We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. Methods and Results We measured PAF-stimulated S-nitrosation of β-catenin and p120-catenin (p120) in three cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein) and CVEC (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. TNF-α increased NO production and caused similar increase in S-nitrosation as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosation of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of NO production and of S-nitrosation blocked PAF-induced S-nitrosation and hyperpermeability whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with VE-cadherin. Conclusions Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability. PMID:22777005

  20. Protein matrix involved in the lipid retention of foie gras during cooking: a multimodal hyperspectral imaging study.

    PubMed

    Théron, Laëtitia; Vénien, Annie; Jamme, Frédéric; Fernandez, Xavier; Peyrin, Frédéric; Molette, Caroline; Dumas, Paul; Réfrégiers, Matthieu; Astruc, Thierry

    2014-06-25

    Denaturation of the protein matrix during heat treatment of duck foie gras was studied in relationship to the amount of fat loss during cooking. A low fat loss group was compared with a high fat loss group by histochemistry, FT-IR, and synchrotron UV microspectroscopy combination to characterize their protein matrix at different scales. After cooking, the high fat loss group showed higher densification of its matrix, higher ultraviolet tyrosine autofluorescence, and an infrared shift of the amide I band. These results revealed a higher level of protein denaturation and aggregation during cooking in high fat loss than in low fat loss foie gras. In addition, the fluorescence and infrared responses of the raw tissue revealed differences according to the level of fat losses after cooking. These findings highlight the importance of the supramolecular state of the protein matrix in determining the fat loss of foie gras.

  1. Long polar fimbriae of enterohemorrhagic Escherichia coli O157:H7 bind to extracellular matrix proteins.

    PubMed

    Farfan, Mauricio J; Cantero, Lidia; Vidal, Roberto; Botkin, Douglas J; Torres, Alfredo G

    2011-09-01

    Adherence to intestinal cells is a key process in infection caused by enterohemorrhagic Escherichia coli (EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract.

  2. Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis.

    PubMed

    Naba, Alexandra; Clauser, Karl R; Hynes, Richard O

    2015-07-23

    The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

  3. Collagen type I may influence the expression of E-cadherin and beta-catenin in carcinoma ex-pleomorphic adenoma.

    PubMed

    Araújo, Vera C; Demasi, Ana Paula Dias; Furuse, Cristiane; Altemani, Albina; Alves, Venâncio A; Freitas, Leandro L; Araújo, Ney S

    2009-07-01

    Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial, but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

  4. The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments

    PubMed Central

    Mitra, Ruchira; Baviskar, Pradyumna; Duncan-Decocq, Rebecca R.; Patel, Darshna

    2012-01-01

    An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding. PMID:22318136

  5. Evidence for ubiquitin-regulated nuclear and subnuclear trafficking among Paramyxovirinae matrix proteins.

    PubMed

    Pentecost, Mickey; Vashisht, Ajay A; Lester, Talia; Voros, Tim; Beaty, Shannon M; Park, Arnold; Wang, Yao E; Yun, Tatyana E; Freiberg, Alexander N; Wohlschlegel, James A; Lee, Benhur

    2015-03-01

    The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear

  6. Adhesive and Signaling Functions of Cadherins and Catenins in Vertebrate Development

    PubMed Central

    Stepniak, Ewa; Radice, Glenn L.; Vasioukhin, Valeri

    2009-01-01

    Properly regulated intercellular adhesion is critical for normal development of all metazoan organisms. Adherens junctions play an especially prominent role in development because they link the adhesive function of cadherin–catenin protein complexes to the dynamic forces of the actin cytoskeleton, which helps to orchestrate a spatially confined and very dynamic assembly of intercellular connections. Intriguingly, in addition to maintaining intercellular adhesion, cadherin–catenin proteins are linked to several major developmental signaling pathways crucial for normal morphogenesis. In this article we will highlight the key genetic studies that uncovered the role of cadherin–catenin proteins in vertebrate development and discuss the potential role of these proteins as molecular biosensors of external cellular microenvironment that may spatially confine signaling molecules and polarity cues to orchestrate cellular behavior throughout the complex process of normal morphogenesis. PMID:20066120

  7. Disturbances of T-cell interactions with endothelium and the extracellular matrix proteins in a patient with Takayasu arteritis.

    PubMed

    Imiela, J; Górski, A; Dybowska, B; Małecki, R; Nosarzewski, J

    1994-06-01

    T-cell interactions with endothelium and the extracellular matrix proteins were studied in vitro in a patient with Takayasu arteritis. Markedly enhanced spontaneous adhesiveness to cultured endothelium and collagen IV and fibronectin were found to be paralleled by abolished responses to the costimulating action of collagens I and IV and fibronectin. Those abnormalities were partly corrected by immunosuppressive therapy. Aberrant interactions of T cells with endothelium and the extracellular matrix proteins may underlay the pathogenesis of some forms of vasculitis. PMID:7911506

  8. Matrix sublimation/recrystallization for imaging proteins by mass spectrometry at high spatial resolution.

    PubMed

    Yang, Junhai; Caprioli, Richard M

    2011-07-15

    We have employed matrix deposition by sublimation for protein image analysis on tissue sections using a hydration/recrystallization process that produces high-quality MALDI mass spectra and high-spatial-resolution ion images. We systematically investigated different washing protocols, the effect of tissue section thickness, the amount of sublimated matrix per unit area, and different recrystallization conditions. The results show that an organic solvent rinse followed by ethanol/water rinses substantially increased sensitivity for the detection of proteins. Both the thickness of the tissue section and the amount of sinapinic acid sublimated per unit area have optimal ranges for maximal protein signal intensity. Ion images of mouse and rat brain sections at 50, 20, and 10 μm spatial resolution are presented and are correlated with hematoxylin and eosin (H&E)-stained optical images. For targeted analysis, histology-directed imaging can be performed using this protocol where MS analysis and H&E staining are performed on the same section.

  9. The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

    PubMed

    Lord, Megan S; Cheng, Bill; McCarthy, Simon J; Jung, MoonSun; Whitelock, John M

    2011-10-01

    Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

  10. Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

    PubMed

    Yonekura, Tomoko; Homma, Hiromi; Sakurai, Atsuo; Moriguchi, Mitsuko; Miake, Yasuo; Toyosawa, Satoru; Shintani, Seikou

    2013-12-01

    Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

  11. A Lipocalin-Derived Peptide Modulating Fibroblasts and Extracellular Matrix Proteins

    PubMed Central

    Carrijo-Carvalho, Linda Christian; Maria, Durvanei A.; Ventura, Janaina S.; Morais, Kátia L. P.; Melo, Robson L.; Rodrigues, Consuelo Junqueira; Chudzinski-Tavassi, Ana Marisa

    2012-01-01

    Lipocalin family members have been implicated in development, regeneration, and pathological processes, but their roles are unclear. Interestingly, these proteins are found abundant in the venom of the Lonomia obliqua caterpillar. Lipocalins are β-barrel proteins, which have three conserved motifs in their amino acid sequence. One of these motifs was shown to be a sequence signature involved in cell modulation. The aim of this study is to investigate the effects of a synthetic peptide comprising the lipocalin sequence motif in fibroblasts. This peptide suppressed caspase 3 activity and upregulated Bcl-2 and Ki-67, but did not interfere with GPCR calcium mobilization. Fibroblast responses also involved increased expression of proinflammatory mediators. Increase of extracellular matrix proteins, such as collagen, fibronectin, and tenascin, was observed. Increase in collagen content was also observed in vivo. Results indicate that modulation effects displayed by lipocalins through this sequence motif involve cell survival, extracellular matrix remodeling, and cytokine signaling. Such effects can be related to the lipocalin roles in disease, development, and tissue repair. PMID:22737165

  12. Structure and expression of an unusually acidic matrix protein of pearl oyster shells.

    PubMed

    Tsukamoto, Daiki; Sarashina, Isao; Endo, Kazuyoshi

    2004-08-01

    We report identification and characterization of the unusually acidic molluscan shell matrix protein Aspein, which may have important roles in calcium carbonate biomineralization. The Aspein gene (aspein) encodes a sequence of 413 amino acids, including a high proportion of Asp (60.4%), Gly (16.0%), and Ser (13.2%), and the predicted isoelectric point is 1.45; this is the most acidic of all the molluscan shell matrix proteins sequenced so far, or probably even of all known proteins on earth. The main body of Aspein is occupied by (Asp)(2-10) sequences punctuated with Ser-Gly dipeptides. RT-PCR demonstrated that the transcript of aspein is expressed at the outer edge of the mantle, corresponding to the calcitic prismatic layer, but not at the inner part of the mantle, corresponding to the aragonitic nacreous layer. Our findings and previous in vitro experiments taken together suggest that Aspein is responsible for directed formation of calcite in the shell of the pearl oyster Pinctada fucata.

  13. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei.

    PubMed

    Gualdrón-López, Melisa; Michels, Paul A M

    2013-02-01

    Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M(r) of 100kDa and 72kDa. 5'-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72kDa. However, recombinant PEX5 migrates aberrantly in SDS-PAGE with an apparent M(r) of 100kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and (35)S-labelled PEX5 showed truncation of the 100kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  14. Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

    PubMed

    Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2004-10-01

    Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

  15. Response of Inflammatory Mediators, Extracellular Matrix Proteins and Stem and Progenitor Cells to Emphysema.

    PubMed

    Skurikhin, E G; Pakhomova, A V; Krupin, V A; Pershina, O V; Pan, E S; Ermolaeva, L A; Vaizova, O E; Rybalkina, O Yu; Dygai, A M

    2016-08-01

    Inflammation, extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, and fibronectin), stem and progenitor cells (multipotent mesenchymal stromal cells, Clara cells, angiogenesis, precursors, endothelial and epithelial cells) were studied in female C57Bl/6 mice with experimental elastase-induced emphysema. Diffuse emphysema reduced the number of endothelial (CD45(-)CD31(+)CD34(+)) and epithelial (CD45(-)CD117(+)CD49f(+)) cells, induced microcirculation disturbances, and decreased the area occupied by the connective tissue. Emphysematous changes in the lungs were accompanied by infiltration of the alveolar septa with macrophages and lymphocytes, increase in the serum and lung concentrations of transforming growth factor-β, IL-1β, IL-2, IL-5, IL-10, and IL-13, and lung concentration of IL-17. In the lungs, inflammation was associated with marked increase in the number of multipotent mesenchymal stromal cells CD90(+)CD73(+)CD106(+)CD44(+)) and Clara cells (CD45(-)CD34(-)CD31(-)Sca1(+)) and overexpression of extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, fibronectin) and Clara cells protein. On the other hand, elastase reduced the number of angiogenic precursor cells (CD45(-)CD117(+)Flk1(+)). PMID:27591877

  16. Flowering and genome integrity control by a nuclear matrix protein in Arabidopsis.

    PubMed

    Xu, Yifeng; Gan, Eng-Seng; He, Yuehui; Ito, Toshiro

    2013-01-01

    The matrix attachment regions (MARs) binding proteins could finely orchestrate temporal and spatial gene expression during development. In Arabidopsis, transposable elements (TEs) and TE-like repeat sequences are transcriptionally repressed or attenuated by the coordination of many key players including DNA methyltransferases, histone deacetylases, histone methyltransferases and the siRNA pathway, which help to protect genomic integrity and control multiple developmental processes such as flowering. We have recently reported that an AT-hook nuclear matrix binding protein, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), participates in a histone deacetylation (HDAC) complex to silence TEs and genes containing a TE-like sequence, including AtMu1, FWA and FLOWERING LOCUS C (FLC) in Ler background. We have shown that TEK knockdown causes increased histone acetylation, reduced H3K9me2 and moderate reduction of DNA methylation in the target loci, leading to the de-repression of FLC and FWA, as well as TE reactivation. Here we discuss the role of TEK as a putative MAR binding protein which functions in the maintenance of genome integrity and in flowering control by silencing TEs and repeat-containing genes. PMID:23836195

  17. Screening of a protein that interacts with the matrix attachment region-binding protein from Dunaliella salina.

    PubMed

    Yang, Rui; Li, Zhaoxi; Lin, Yan; Yang, Baosheng; Wang, Tianyun

    2013-01-01

    We isolated the matrix attachment region-binding protein (MBP) DMBP-1 from Dunaliella salina in our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening the D. salina cDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays.

  18. 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.

  19. Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

    PubMed

    Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

    2013-11-01

    Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

  20. A chondroitin sulfate chain attached to the bone dentin matrix protein 1 NH2-terminal fragment.

    PubMed

    Qin, Chunlin; Huang, Bingzhen; Wygant, James N; McIntyre, Bradley W; McDonald, Charles H; Cook, Richard G; Butler, William T

    2006-03-24

    Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein shown by gene ablations to be critical for the proper mineralization of bone and dentin. In the extracellular matrix of these tissues DMP1 is present as fragments representing the NH2-terminal (37 kDa) and COOH-terminal (57 kDa) portions of the cDNA-deduced amino acid sequence. During our separation of bone noncollagenous proteins, we observed a high molecular weight, DMP1-related component (designated DMP1-PG). We purified DMP1-PG with a monoclonal anti-DMP1 antibody affinity column. Amino acid analysis and Edman degradation of tryptic peptides proved that the core protein for DMP1-PG is the 37-kDa fragment of DMP1. Chondroitinase treatments demonstrated that the slower migration rate of DMP1-PG is due to the presence of glycosaminoglycan. Quantitative disaccharide analysis indicated that the glycosaminoglycan is made predominantly of chondroitin 4-sulfate. Further analysis on tryptic peptides led us to conclude that a single glycosaminoglycan chain is linked to the core protein via Ser74, located in the Ser74-Gly75 dipeptide, an amino acid sequence specific for the attachment of glycosaminoglycans. Our findings show that in addition to its existence as a phosphoprotein, the NH2-terminal fragment from DMP1 occurs as a proteoglycan. Amino acid sequence alignment analysis showed that the Ser74-Gly75 dipeptide and its flanking regions are highly conserved among a wide range of species from caiman to the Homo sapiens, indicating that this glycosaminoglycan attachment domain has survived an extremely long period of evolution pressure, suggesting that the glycosaminoglycan may be critical for the basic biological functions of DMP1.

  1. Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus

    PubMed Central

    Förster, Andreas; Maertens, Goedele N.; Farrell, Paul J.

    2015-01-01

    ABSTRACT Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect the conformation of the fusion protein, the major current target for RSV antivirals, in the mature virus. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize, and biochemical data suggest that RSV M also dimerizes. Here, using size exclusion chromatography-multiangle laser light scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutations destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, budding and release of virus-like particles are prevented in M mutants that fail to form filaments. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher-order oligomers triggers viral filament assembly and virus production. IMPORTANCE Human respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and antiviral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular

  2. The Spindle Matrix Protein, Chromator, Is a Novel Tubulin Binding Protein That Can Interact with Both Microtubules and Free Tubulin

    PubMed Central

    Yao, Changfu; Wang, Chao; Li, Yeran; Ding, Yun; Rath, Uttama; Sengupta, Saheli; Girton, Jack; Johansen, Kristen M.; Johansen, Jørgen

    2014-01-01

    The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs. PMID:25072297

  3. Stimulation of Periodontal Ligament Stem Cells by Dentin Matrix Protein 1 Activates Mitogen-Activated Protein Kinase and Osteoblast Differentiation

    PubMed Central

    Chandrasekaran, Sangeetha; Ramachandran, Amsaveni; Eapen, Asha; George, Anne

    2013-01-01

    Background Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). Methods hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal-regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. Results Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase-2, alkaline phosphatase, and transforming growth factor β1. Activation of ERK mitogen-activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1-treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. Conclusion DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration. PMID:22612367

  4. Investigation of the molecular mechanism of δ-catenin ubiquitination: Implication of β-TrCP-1 as a potential E3 ligase.

    PubMed

    Shrestha, Hridaya; Yuan, Tingting; He, Yongfeng; Moon, Pyong-Gon; Shrestha, Nensi; Ryu, Taeyong; Park, So-Yeon; Cho, Young-Chang; Lee, Chan-Hyeong; Baek, Moon-Chang; Cho, Sayeon; Simkhada, Shishli; Kim, Hangun; Kim, Kwonseop

    2016-09-01

    Ubiquitination, a post-translational modification, involves the covalent attachment of ubiquitin to the target protein. The ubiquitin-proteasome pathway and the endosome-lysosome pathway control the degradation of the majority of eukaryotic proteins. Our previous study illustrated that δ-catenin ubiquitination occurs in a glycogen synthase kinase-3 (GSK-3) phosphorylation-dependent manner. However, the molecular mechanism of δ-catenin ubiquitination is still unknown. Here, we show that the lysine residues required for ubiquitination are located mainly in the C-terminal portion of δ-catenin. In addition, we provide evidence that β-TrCP-1 interacts with δ-catenin and functions as an E3 ligase, mediating δ-catenin ubiquitin-proteasome degradation. Furthermore, we prove that both the ubiquitin-proteasome pathway and the lysosome degradation pathway are involved in δ-catenin degradation. Our novel findings on the mechanism of δ-catenin ubiquitination will add a new perspective to δ-catenin degradation and the effects of δ-catenin on E-cadherin involved in epithelial cell-cell adhesion, which is implicated in prostate cancer progression.

  5. OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

    PubMed Central

    Fernandes, Luis G. V.; Vieira, Monica L.; Kirchgatter, Karin; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Romero, Eliete C.

    2012-01-01

    Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection. PMID:22802342

  6. The Extracellular Matrix of Staphylococcus aureus Biofilms Comprises Cytoplasmic Proteins That Associate with the Cell Surface in Response to Decreasing pH

    PubMed Central

    Foulston, Lucy; Elsholz, Alexander K. W.; DeFrancesco, Alicia S.

    2014-01-01

    ABSTRACT Biofilm formation by Staphylococcus aureus involves the formation of an extracellular matrix, but the composition of this matrix has been uncertain. Here we report that the matrix is largely composed of cytoplasmic proteins that reversibly associate with the cell surface in a manner that depends on pH. We propose a model for biofilm formation in which cytoplasmic proteins are released from cells in stationary phase. These proteins associate with the cell surface in response to decreasing pH during biofilm formation. Rather than utilizing a dedicated matrix protein, S. aureus appears to recycle cytoplasmic proteins that moonlight as components of the extracellular matrix. PMID:25182325

  7. Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling

    PubMed Central

    Chairoungdua, Arthit; Smith, Danielle L.; Pochard, Pierre; Hull, Michael

    2010-01-01

    CD82 and CD9 are tetraspanin membrane proteins that can function as suppressors of tumor metastasis. Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin–mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels. Inhibition of Wnt/β-catenin signaling is independent of glycogen synthase kinase-3β and of the proteasome- and lysosome-mediated protein degradation pathways. CD82 and CD9 expression induces β-catenin export via exosomes, which is blocked by a sphingomyelinase inhibitor, GW4869. CD82 fails to induce exosome release of β-catenin in cells that express low levels of E-cadherin. Exosome release from dendritic cells generated from CD9 knockout mice is reduced compared with that from wild-type dendritic cells. These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin. Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways. PMID:20837771

  8. Tetrandrine Inhibits the Wnt/β-Catenin Signalling Pathway and Alleviates Osteoarthritis: An In Vitro and In Vivo Study

    PubMed Central

    Zhou, Xindie; Li, Weijun; Jiang, Lifeng; Bao, Jiapeng; Tao, Lijiang; Li, Jin; Wu, Lidong

    2013-01-01

    There is currently no effective drug treatment for the early phase of osteoarthritis (OA), one of the most common senile diseases. The goal of this study was to investigate the protective effect of the tetrandrine (Tet) on OA, in vitro and in vivo. In an in vitro experiment, quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate changes in gene expression upon the addition of Tet in chondrocytes processed with IL-1β; changes in protein profiles were assessed by Western blotting. In vivo, to determine whether Tet has the protective effects on articular cartilage, a rabbit anterior cruciate ligament transaction model of OA was established. Expression of matrix metalloproteinase and β-catenin genes increased significantly, while that of tissue inhibitor of metalloproteinase-1 decreased significantly in the OA group both in vivo and in chondrocytes. However, the changes of expression were reversed by Tet, and there was less cartilage degradation in vivo compared with the OA group, as assessed by histological and macroscopic observations. Thus, Tet may play a useful role in the treatment of OA through the Wnt/β-catenin signalling pathway and has potential for the treatment of OA. PMID:23533523

  9. Tetrandrine Inhibits the Wnt/ β -Catenin Signalling Pathway and Alleviates Osteoarthritis: An In Vitro and In Vivo Study.

    PubMed

    Zhou, Xindie; Li, Weijun; Jiang, Lifeng; Bao, Jiapeng; Tao, Lijiang; Li, Jin; Wu, Lidong

    2013-01-01

    There is currently no effective drug treatment for the early phase of osteoarthritis (OA), one of the most common senile diseases. The goal of this study was to investigate the protective effect of the tetrandrine (Tet) on OA, in vitro and in vivo. In an in vitro experiment, quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate changes in gene expression upon the addition of Tet in chondrocytes processed with IL-1 β ; changes in protein profiles were assessed by Western blotting. In vivo, to determine whether Tet has the protective effects on articular cartilage, a rabbit anterior cruciate ligament transaction model of OA was established. Expression of matrix metalloproteinase and β -catenin genes increased significantly, while that of tissue inhibitor of metalloproteinase-1 decreased significantly in the OA group both in vivo and in chondrocytes. However, the changes of expression were reversed by Tet, and there was less cartilage degradation in vivo compared with the OA group, as assessed by histological and macroscopic observations. Thus, Tet may play a useful role in the treatment of OA through the Wnt/ β -catenin signalling pathway and has potential for the treatment of OA. PMID:23533523

  10. Structural similarity between the p17 matrix protein of HIV-1 and interferon-gamma.

    PubMed

    Matthews, S; Barlow, P; Boyd, J; Barton, G; Russell, R; Mills, H; Cunningham, M; Meyers, N; Burns, N; Clark, N

    1994-08-25

    The human immunodeficiency virus (HIV) matrix protein, p17, forms the outer shell of the core of the virus, lining the inner surface of the viral membrane. The protein has several key functions. It orchestrates viral assembly via targeting signals that direct the gag precursor polyprotein, p55, to the host cell membrane and it interacts with the transmembrane protein, gp41, to retain the env-encoded proteins in the virus. In addition, p17 contains a nuclear localization signal that directs the preintegration complex to the nucleus of infected cells. This permits the virus to infect productively non-dividing cells, a distinguishing feature of HIV and other lentiviruses. We have determined the solution structure of p17 by nuclear magnetic resonance (NMR) with a root-mean square deviation for the backbone of the well-defined regions of 0.9 A. It consists of four helices connected by short loops and an irregular, mixed beta-sheet which provides a positively charged surface for interaction with the inner layer of the membrane. The helical topology is unusual; the Brookhaven protein database contains only one similar structure, that of the immune modulator interferon-gamma.

  11. Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

    NASA Astrophysics Data System (ADS)

    Rusen, L.; Mustaciosu, C.; Mitu, B.; Filipescu, M.; Dinescu, M.; Dinca, V.

    2013-08-01

    Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm-2. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

  12. Expression and deposition of fibrous extracellular matrix proteins in cardiac valves during chick development.

    PubMed

    Tan, Hong; Junor, Lorain; Price, Robert L; Norris, Russell A; Potts, Jay D; Goodwin, Richard L

    2011-02-01

    Extracellular matrix (ECM) plays essential signaling and structural roles required for the proper function of cardiac valves. Cardiac valves initially form as jelly-like cushions, which must adapt to withstand the increased circulation hemodynamics associated with fetal development and birth. This increased biomechanical stability of the developing valves is largely imparted by ECM proteins, which form a highly organized fibrous meshwork. Since heart valve defects contribute to most congenital heart diseases, understanding valve development will provide insight into the pathogenesis of various congenital valve anomalies. Thus, the goal of this study is to describe the spatiotemporal deposition of fibrous ECM proteins during cardiac valve development. Chick embryonic and fetal atrioventricular and semilunar valves were examined by light, confocal, and transmission electron microscopy (TEM). Our data demonstrate that fibrous ECM proteins are deposited when the leaflets are adopting an elongated and compacted phenotype. A general pattern of increased fibrotic ECM deposition was detected in valve tissues. Also, each ECM protein examined displayed a unique pattern of organization, suggesting that regulation of fibrous protein deposition is complex and likely involves both genetic and mechanical factors. In addition, the TEM study revealed the presence of membrane protrusions from valvular endocardium, indicating a potential mechanism for mechanical force transduction.

  13. Characterization of a putative extracellular matrix protein from the beetle Tenebrio molitor: hormonal regulation during metamorphosis.

    PubMed

    Royer, Véronique; Hourdry, Alban; Fraichard, Stéphane; Bouhin, Hervé

    2004-03-01

    We used differential display to isolate epidermis cDNAs corresponding to juvenile-hormone analog-regulated mRNA from the beetle Tenebrio molitor. One of them encodes a putative extracellular matrix (ECM) protein, named Tenebrin. Indeed, the deduced protein sequence contains ECM typical features like the presence of a signal peptide, internal repeats, a RGD tripeptide sequence motif known to bind integrins and von Willebrand factor type c domains involved in protein-protein interactions. Northern blot analysis reveals a single transcript of about 11 kb with an expression pattern correlated to 20-hydroxyecdysone fluctuations during metamorphosis. In vivo injections of exogenous 20-hydroxyecdysone alone or combined with cycloheximide show that Tenebrin expression is directly induced by this hormone. Methoprene (a juvenile hormone analog) application experiments show that Tenebrin expression is rapidly induced by this analog. This gene is still up-regulated in the presence of protein synthesis inhibitor but, in these conditions, the mRNA induction level is not maximal.

  14. Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction.

    PubMed

    Singhal, Neha; Martin, Paul T

    2011-11-01

    The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission. PMID:21766463

  15. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis.

    PubMed

    Tian, Yuan; Mok, Myth T S; Yang, Pengyuan; Cheng, Alfred S L

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. PMID:27556491

  16. Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin.

    PubMed

    Provost, Elayne; McCabe, Anthony; Stern, Jessica; Lizardi, Isabel; D'Aquila, Thomas G; Rimm, David L

    2005-04-14

    Beta-catenin is a multifunctional protein involved in both cadherin-mediated adhesion and the wnt signaling cascade. Mutations in exon 3 of beta-catenin have been identified in many cancers. In addition to disruption of key serine and threonine residues, mutations are frequently reported in other residues in exon 3 that are not kinase substrates. The most frequently mutated nonserine/threonine residues are D32 and G34. Since D32 and G34 are part of the ubiquitination destruction motif, DSGPhiXS, we hypothesize that this motif may contribute to disruption of beta-catenin homeostasis and lead to cellular transformation. We demonstrate that the mutants D32A and G34A exhibit no change in phosphorylation by GSK3beta, but display reduced ubiquitination compared to wild-type and S33A mutant beta-catenin. To assess the functional implications of these mutations, we created stable MDCK cell lines expressing these constructs. We found that stable cell lines harboring D32A-mutated beta-catenin were highly transformed, while S33A and G34 demonstrated only weak transforming properties in our assays. Despite altered ubiquitination status and increased transformation, the D32A mutant cell line does not display transcriptional activation of standard target genes. Therefore, D32A mutation may mediate transformation by an alternative beta-catenin-mediated signaling pathway.

  17. [Effect of phenylhexyl isothiocyanate on Wnt/beta-catenin signaling pathway in Jurkat cell line].

    PubMed

    Lin, Juan; Huang, Yi-Qun; Ma, Xu-Dong

    2013-04-01

    This study was purposed to investigate the effect of phenylhexyl isothiocyanate (PHI) on Wnt/β-catenin signaling pathway, histone acetylation, histone methylation and cell apoptosis in Jurkat cell line. The viability of Jurkat cells after treatment with PHI was tested by MTT. Apoptotic rate of Jurkat cells was measured by flow cytometry. The levels of Wnt/β-catenin related proteins including β-catenin, TCF, c-myc, and cyclinD1, histone acetylated H3 and H4, histone methylated H3K9 and H3K4 were detected by Western blot. The results showed that PHI inhibited the cell growth and induced apoptosis in Jurkat cells in time-and dose-dependent manners. Its IC50 at 48 h was about 20 µmol/L. Expression of histone acetylated H3, H4 and histone methylated H3k4 increased after exposure to PHI for 3 h, while histone methylated H3K9 decreased. Expression of β-catenin was not changed after exposure to PHI for 3 h, but expression of β-catenin, and its cell cycle-related genes such as TCF, c-myc and cyclinD1 decreased after exposure to PHI for 7 h. It is concluded that PHI regulates acetylation and methylation of histone, inhibits Wnt/β-catenin signal pathway, and is able to induce apoptosis and inhibits growth of Jurkat cells. PMID:23628033

  18. β-Catenin/Tcf signaling in murine oocytes identifies nonovulatory follicles.

    PubMed

    Usongo, Macalister; Rizk, Aida; Farookhi, Riaz

    2012-12-01

    WNTS are secreted glycoprotein molecules that signal through one of three signaling pathways. The best-characterized pathway involves stabilization of the multifunctional protein β-catenin, which in concert with members of the T-cell factor (Tcf) family activates specific gene transcription. We have examined putative Wnt/β-catenin in the murine ovary using transgenic mice harboring a reporter construct that activates β-galactosidase (lacZ) expression in response to β-catenin/Tcf binding (TopGal mice). Primordial and primary follicles did not stain for lacZ, and the proportion of β-catenin/Tcf signaling oocytes was lower than that of nonsignaling oocytes throughout estrous cycle. β-Catenin/Tcf signaling oocytes were observed in follicles from the secondary stage of development and their proportion increased with follicular maturation (secondary follicles, 20%; early antral and antral follicles, 70%). In contrast, the majority (>90%) of ovulated oocytes did not stain for lacZ. As the oocyte possesses components for WNT signal transduction, our data suggest that β-catenin/Tcf signaling is involved in the development of follicular ovulatory capability and identifies nonovulatory follicles. PMID:23006471

  19. BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

    PubMed Central

    Zhang, Qiangling; Shi, Xin-E; Song, Chengchuang; Sun, Shiduo; Yang, Gongshe; Li, Xiao

    2015-01-01

    Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI) is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway. PMID:26247931

  20. Palmitoylation of δ-catenin by DHHC5 Mediates Activity-Induced Synapse Plasticity

    PubMed Central

    Brigidi, G. Stefano; Sun, Yu; Beccano-Kelly, Dayne; Pitman, Kimberley; Mobasser, Mahsan; Borgland, Stephanie L.; Milnerwood, Austen J.; Bamji, Shernaz X.

    2016-01-01

    Synaptic cadherin adhesion complexes are known to be key regulators of synapse plasticity. However, the molecular mechanisms that coordinate activity-induced modifications in cadherin localization and adhesion and subsequent changes in synapse morphology and efficacy, remain unanswered. We demonstrate that the intracellular cadherin binding protein, δ-catenin, is transiently palmitoylated by DHHC5 following enhanced synaptic activity, and that palmitoylation increases δ-catenin/cadherin interactions at synapses. Both the palmitoylation of δ-catenin and its binding to cadherin are required for activity-induced stabilization of N-cadherin at synapses, the enlargement of postsynaptic spines, as well as insertion of GluA1 and GluA2 subunits into the synaptic membrane and the concomitant increase in mEPSC amplitude. Importantly, context-dependent fear conditioning in mice results in increased δ-catenin palmitoylation as well as increased δ-catenin/cadherin associations at hippocampal synapses. Together, this suggests a role for palmitoylated δ-catenin in coordinating activity-dependent changes in synaptic adhesion molecules, synapse structure, and receptor localization that are involved in memory formation. PMID:24562000

  1. Epigenetic Activation of Wnt/β-Catenin Signaling in NAFLD-Associated Hepatocarcinogenesis

    PubMed Central

    Tian, Yuan; Mok, Myth T.S.; Yang, Pengyuan; Cheng, Alfred S.L.

    2016-01-01

    Non-alcoholic fatty liver disease (NAFLD), characterized by fat accumulation in liver, is closely associated with central obesity, over-nutrition and other features of metabolic syndrome, which elevate the risk of developing hepatocellular carcinoma (HCC). The Wnt/β-catenin signaling pathway plays a significant role in the physiology and pathology of liver. Up to half of HCC patients have activation of Wnt/β-catenin signaling. However, the mutation frequencies of CTNNB1 (encoding β-catenin protein) or other antagonists targeting Wnt/β-catenin signaling are low in HCC patients, suggesting that genetic mutations are not the major factor driving abnormal β-catenin activities in HCC. Emerging evidence has demonstrated that obesity-induced metabolic pathways can deregulate chromatin modifiers such as histone deacetylase 8 to trigger undesired global epigenetic changes, thereby modifying gene expression program which contributes to oncogenic signaling. This review focuses on the aberrant epigenetic activation of Wnt/β-catenin in the development of NAFLD-associated HCC. A deeper understanding of the molecular mechanisms underlying such deregulation may shed light on the identification of novel druggable epigenetic targets for the prevention and/or treatment of HCC in obese and diabetic patients. PMID:27556491

  2. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    PubMed

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  3. Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins.

    PubMed

    Bosshardt, Dieter D; Sculean, Anton; Donos, Nikolaos; Lang, Niklaus P

    2006-05-01

    A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern. PMID:16674690

  4. Induction of Wnt/β-Catenin Signaling in Mouse Mesenchymal Stem Cells Is Associated with Activation of the p130 and E2f4 and Formation of the p130/Gsk3β/β-Catenin Complex

    PubMed Central

    Petrov, Nikolay; Zhidkova, Olga; Serikov, Vladimir; Zenin, Valery

    2012-01-01

    Proteins p130 and E2f4, members of the retinoblastoma protein (pRb) family/E2F transcription factor family, are the key elements in regulation of cell cycle and differentiation. The functional role of the p130/E2f4 in mesenchymal stem cells (MSC) is unclear. We demonstrate here that activation of the Wnt/β-catenin pathway in mouse MSC is associated with accumulation of active forms of the p130, E2f4, and β-catenin but does not result in inhibition of cell cycle progression. The levels and phosphorylation patterns of p130, E2f4, and β-catenin in MSC do not change during cell cycle progression. This is different from control T98G glyoblastoma cells that accumulated differently phosphorylated forms of the p130 in quiescence, and under active proliferation. In MSC, synchronized at G0/G1 and S cell cycle phases, the p130 and β-catenin physically interact each other, whereas Gsk3β was associated and co-precipitated with both p130 and β-catenin. Our results indicate that Wnt/β-catenin and pRb signal pathways interact with each other and form common p130/Gsk3β/β-catenin complex during MSC cycle progression. Physiological relevance of such complex may be associated with coupling of the cell cycle and differentiation in MSC, which is related to a wide differentiation potential of these stem cells. PMID:21631154

  5. β-Catenin/LEF1 transactivates the microRNA-371-373 cluster that modulates the Wnt/β-catenin-signaling pathway.

    PubMed

    Zhou, A-D; Diao, L-T; Xu, H; Xiao, Z-D; Li, J-H; Zhou, H; Qu, L-H

    2012-06-14

    The microRNA-371-373 (miR-371-373) cluster is specifically expressed in human embryonic stem cells (ESCs) and is thought to be involved in stem cell maintenance. Recently, microRNAs (miRNAs) of this cluster were shown to be frequently upregulated in several human tumors. However, the regulatory mechanism for the involvement of the miR-371-373 cluster in human ESCs or cancer cells remains unclear. In this study, we explored the relationship between this miRNA cluster and the Wnt/β-catenin-signaling pathway, which has been shown to be involved in both stem cell maintenance and tumorigenesis. We show that miR-371-373 expression is induced by lithium chloride and is positively correlated with Wnt/β-catenin-signaling activity in several human cancer cell lines. Mechanistically, three TCF/LEF1-binding elements (TBEs) were identified in the promoter region and shown to be required for Wnt-dependent activation of miR-371-373. Interestingly, we also found that miR-372&373, in turn, activate Wnt/β-catenin signaling. In addition, four protein genes related to the Wnt/β-catenin-signaling pathway were identified as direct targets of miR-372&373, including Dickkopf-1 (DKK1), a well-known inhibitor of Wnt/β-catenin signaling. Using a lentiviral system, we showed that overexpression of miR-372 or miR-373 promotes cell growth and the invasive activity of tumor cells as knockdown of DKK1. Taken together, our study demonstrates a novel β-catenin/LEF1-miR-372&373-DKK1 regulatory feedback loop, which may have a critical role in regulating the activity of Wnt/β-catenin signaling in human cancer cells.

  6. Bone-related matrix proteins expression in vitro and in vivo by marrow stromal cell line.

    PubMed

    Benayahu, D; Gurevitz, O A; Shamay, A

    1994-10-01

    MBA-1, a bone marrow stroma-derived cell line, was transplanted in an ectopic site and formed endochondral bone. The ossicle developed through stages of cell proliferation, differentiated into a zone of hypertrophy and formed a chondroid-like area which further developed to primary mineralized bone. We explored the expression of various matrix proteins by MBA-1 cells in vitro and in the ossicle formed in vivo. MBA-1 cells constitutively expressed mRNAs encoding for collagen I, non-collagenous proteins and alkaline phosphatase. RNA extracted from the ossicle formed by these cells was expressed in a different pattern. The in vivo maturation of MBA-1 cells was accompanied by low expression of mRNA for procollagen alpha 2(I) and a marked increase in osteonectin and osteopontin mRNA levels. Thus, the ability to follow expression of these genes through bone formation in vivo has been demonstrated. PMID:9437244

  7. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    PubMed Central

    Gannagé, Monique; Schmid, Dorothee; Albrecht, Randy; Dengjel, Jörn; Torossi, Tania; Rämer, Patrick C.; Lee, Monica; Strowig, Till; Arrey, Frida; Conenello, Gina; Pypaert, Marc; Andersen, Jens; García-Sastre, Adolfo; Münz, Christian

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here we demonstrate that live influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes. Matrix protein 2 is sufficient and necessary for this inhibition of autophagosome degradation. Macroautophagy inhibition compromises cell survival of influenza virus infected cells, but does not influence viral replication. We propose that influenza A virus, which also encodes pro-apoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and blocking macroautophagy. PMID:19837376

  8. 5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing β-catenin.

    PubMed

    Fatima, Sarwat; Shi, Xiaoke; Lin, Zesi; Chen, Guo-qing; Pan, Xiao-hua; Wu, Justin Che-Yuen; Ho, John W; Lee, Nikki P; Gao, Hengjun; Zhang, Ge; Lu, Aiping; Bian, Zhao Xiang

    2016-02-01

    5-Hydroxytryptamine (5-HT), a neurotransmitter and vasoactive factor, has been reported to promote proliferation of serum-deprived hepatocellular carcinoma (HCC) cells but the detailed intracellular mechanism is unknown. As Wnt/β-catenin signalling is highly dysregulated in a majority of HCC, this study explored the regulation of Wnt/β-catenin signalling by 5-HT. The expression of various 5-HT receptors was studied by quantitative real-time polymerase chain reaction (qPCR) in HCC cell lines as well as in 33 pairs of HCC tumours and corresponding adjacent non-tumour tissues. Receptors 5-HT1D (21/33, 63.6%), 5-HT2B (12/33, 36.4%) and 5-HT7 (15/33, 45.4%) were overexpressed whereas receptors 5-HT2A (17/33, 51.5%) and 5-HT5 (30/33, 90.1%) were reduced in HCC tumour tissues. In vitro data suggests 5-HT increased total β-catenin, active β-catenin and decreased phosphorylated β-catenin protein levels in serum deprived HuH-7 and HepG2 cells compared to control cells under serum free medium without 5-HT. Activation of Wnt/β-catenin signalling was evidenced by increased expression of β-catenin downstream target genes, Axin2, cyclin D1, dickoppf-1 (DKK1) and glutamine synthetase (GS) by qPCR in serum-deprived HCC cell lines treated with 5-HT. Additionally, biochemical analysis revealed 5-HT disrupted Axin1/β-catenin interaction, a critical step in β-catenin phosphorylation. Increased Wnt/β-catenin activity was attenuated by antagonist of receptor 5-HT7 (SB-258719) in HCC cell lines and patient-derived primary tumour tissues in the presence of 5-HT. SB-258719 also reduced tumour growth in vivo. This study provides evidence of Wnt/β-catenin signalling activation by 5-HT and may represent a potential therapeutic target for hepatocarcinogenesis. PMID:26474915

  9. BETA-CATENIN SIGNALING, LIVER REGENERATION AND HEPATOCELLULAR CANCER: SORTING THE GOOD FROM THE BAD

    PubMed Central

    Nejak-Bowen, Kari Nichole; Monga, Satdarshan P. S.

    2011-01-01

    Among the adult organs, liver is unique for its ability to regenerate. A concerted signaling cascade enables optimum initiation of the regeneration process following insults brought about by surgery or a toxicant. Additionally, there exists a cellular redundancy, whereby a transiently amplifying progenitor population appears and expands to ensure regeneration, when differentiated cells of the liver are unable to proliferate in both experimental and clinical scenarios. One such pathway of relevance in these phenomena is Wnt/β-catenin signaling, which is activated relatively early during regeneration mostly through post-translational modifications. Once activated, β-catenin signaling drives the expression of target genes that are critical for cell cycle progression and contribute to initiation of the regeneration process. The role and regulation of Wnt/β-catenin signaling is now documented in rats, mice, zebrafish and patients. More recently, a regenerative advantage of the livers in β-catenin overexpressing mice was reported, as was also the case after exogenous Wnt-1 delivery to the liver paving the way for assessing means to stimulate the pathway for therapeutics in liver failure. β-Catenin is also pertinent in hepatic oval cell activation and differentiation. However, aberrant activation of the Wnt/β-catenin signaling is reported in a significant subset of hepatocellular cancers (HCC). While many mechanisms of such activation have been reported, the most functional means of aberrant and sustained activation is through mutations in the β-catenin gene or in AXIN1/2, which encodes for a scaffolding protein critical for β-catenin degradation. Intriguingly, in experimental models hepatic overexpression of normal or mutant β-catenin is insufficient for tumorigenesis. In fact β-catenin loss promoted chemical carcinogenesis in the liver due to alternate mechanisms. Since most HCC occur in the backdrop of chronic hepatic injury, where hepatic regeneration is

  10. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Ilalov, Kiril; Yu, Shuang; Xu, Ke; Prazak, Lisa; Fajardo, Marc; Sehgal, Bantoo; Di Cesare, Paul E.

    2006-01-01

    Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.—Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J. 20, E129 -E140 (2006) PMID:16585064

  11. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    SciTech Connect

    Gualdrón-López, Melisa; Michels, Paul A.M.

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  12. Identification of full-length dentin matrix protein 1 in dentin and bone.

    PubMed

    Huang, Bingzhen; Maciejewska, Izabela; Sun, Yao; Peng, Tao; Qin, Disheng; Lu, Yongbo; Bonewald, Lynda; Butler, William T; Feng, Jian; Qin, Chunlin

    2008-05-01

    Dentin matrix protein 1 (DMP1) has been identified in the extracellular matrix (ECM) of dentin and bone as the processed NH(2)-terminal and COOH-terminal fragment. However, the full-length form of DMP1 has not been identified in these tissues. The focus of this investigation was to search for the intact full-length DMP1 in dentin and bone. We used two types of anti-DMP1 antibodies to identify DMP1: one type specifically recognizes the NH(2)-terminal region and the other type is only reactive to the COOH-terminal region of the DMP1 amino acid sequence. An approximately 105-kDa protein, extracted from the ECM of rat dentin and bone, was recognized by both types of antibodies; and the migration rate of this protein was identical to the recombinant mouse full-length DMP1 made in eukaryotic cells. We concluded that this approximately 105-kDa protein is the full-length form of DMP1, which is considerably less abundant than its processed fragments in the ECM of dentin and bone. We also detected the full-length form of DMP1 and its processed fragments in the extract of dental pulp/odontoblast complex dissected from rat teeth. In addition, immunofluorescence analysis showed that in MC3T3-E1 cells the NH(2)-terminal and COOH-terminal fragments of DMP1 are distributed differently. Our findings indicate that the majority of DMP1 must be cleaved within the cells that synthesize it and that minor amounts of uncleaved DMP1 molecules are secreted into the ECM of dentin and bone.

  13. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

    PubMed

    Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Yang, Qiwei; Tian, Yufeng; Morales La Madrid, Andres; Mirzoeva, Salida; Bouyer, Patrice G; Xu, David; Walker, Matthew; Cohn, Susan L

    2011-01-01

    Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the

  14. The destruction complex of beta-catenin in colorectal carcinoma and colonic adenoma

    PubMed Central

    Bourroul, Guilherme Muniz; Fragoso, Hélio José; Gomes, José Walter Feitosa; Bourroul, Vivian Sati Oba; Oshima, Celina Tizuko Fujiyama; Gomes, Thiago Simão; Saba, Gabriela Tognini; Palma, Rogério Tadeu; Waisberg, Jaques

    2016-01-01

    ABSTRACT Objective To evaluate the destruction complex of beta-catenin by the expression of the proteins beta-catetenin, adenomatous polyposis coli, GSK3β, axin and ubiquitin in colorectal carcinoma and colonic adenoma. Methods Tissue samples from 64 patients with colorectal carcinoma and 53 patients with colonic adenoma were analyzed. Tissue microarray blocks and slides were prepared and subjected to immunohistochemistry with polyclonal antibodies in carcinoma, adjacent non-neoplastic mucosa, and adenoma tissues. The immunoreactivity was evaluated by the percentage of positive stained cells and by the intensity assessed through of the stained grade of proteins in the cytoplasm and nucleus of cells. In the statistical analysis, the Spearman correlation coefficient, Student’s t, χ2, Mann-Whitney, and McNemar tests, and univariate logistic regression analysis were used. Results In colorectal carcinoma, the expressions of beta-catenin and adenomatous polyposis coli proteins were significantly higher than in colonic adenomas (p<0.001 and p<0.0001, respectively). The immunoreactivity of GSK3β, axin 1 and ubiquitin proteins was significantly higher (p=0.03, p=0.039 and p=0.03, respectively) in colorectal carcinoma than in the colonic adenoma and adjacent non-neoplastic mucosa. The immunohistochemistry staining of these proteins did not show significant differences with the clinical and pathological characteristics of colorectal cancer and colonic adenoma. Conclusions These results suggest that, in adenomas, the lower expression of the beta-catenin, axin 1 and GSK3β proteins indicated that the destruction complex of beta-catenin was maintained, while in colorectal carcinoma, the increased expression of beta-catenin, GSK3β, axin 1, and ubiquitin proteins indicated that the destruction complex of beta-catenin was disrupted. PMID:27462886

  15. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  16. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross

  17. Protein complex detection via weighted ensemble clustering based on Bayesian nonnegative matrix factorization.

    PubMed

    Ou-Yang, Le; Dai, Dao-Qing; Zhang, Xiao-Fei

    2013-01-01

    Detecting protein complexes from protein-protein interaction (PPI) networks is a challenging task in computational biology. A vast number of computational methods have been proposed to undertake this task. However, each computational method is developed to capture one aspect of the network. The performance of different methods on the same network can differ substantially, even the same method may have different performance on networks with different topological characteristic. The clustering result of each computational method can be regarded as a feature that describes the PPI network from one aspect. It is therefore desirable to utilize these features to produce a more accurate and reliable clustering. In this paper, a novel Bayesian Nonnegative Matrix Factorization (NMF)-based weighted Ensemble Clustering algorithm (EC-BNMF) is proposed to detect protein complexes from PPI networks. We first apply different computational algorithms on a PPI network to generate some base clustering results. Then we integrate these base clustering results into an ensemble PPI network, in the form of weighted combination. Finally, we identify overlapping protein complexes from this network by employing Bayesian NMF model. When generating an ensemble PPI network, EC-BNMF can automatically optimize the values of weights such that the ensemble algorithm can deliver better results. Experimental results on four PPI networks of Saccharomyces cerevisiae well verify the effectiveness of EC-BNMF in detecting protein complexes. EC-BNMF provides an effective way to integrate different clustering results for more accurate and reliable complex detection. Furthermore, EC-BNMF has a high degree of flexibility in the choice of base clustering results. It can be coupled with existing clustering methods to identify protein complexes.

  18. Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

    PubMed Central

    Camper, Lisbet; Heinegård, Dick; Lundgren-Åkerlund, Evy

    1997-01-01

    Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg2+ or Mn2+ but not Ca2+. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ∼110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading. PMID:9281592

  19. An ensemble method with hybrid features to identify extracellular matrix proteins.

    PubMed

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc.

  20. Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells.

    PubMed

    Celebi, Betül; Mantovani, Diego; Pineault, Nicolas

    2011-10-01

    Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein.

  1. Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas

    PubMed Central

    Bognar, M K; Vincendeau, M; Erdmann, T; Seeholzer, T; Grau, M; Linnemann, J R; Ruland, J; Scheel, C H; Lenz, P; Ott, G; Lenz, G; Hauck, S M; Krappmann, D

    2016-01-01

    Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis. PMID:26776161

  2. Inhibition of β-Catenin to Overcome Endocrine Resistance in Tamoxifen-Resistant Breast Cancer Cell Line

    PubMed Central

    Won, Hye Sung; Lee, Kyung Mee; Oh, Ju Eon; Nam, Eun Mi; Lee, Kyoung Eun

    2016-01-01

    Background The β-catenin signaling is important in cell growth and differentiation and is frequently dysregulated in various cancers. The most well-known mechanism of endocrine resistance is cross-talk between the estrogen receptor (ER) and other growth factor signaling, such as phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway. In the present study, we investigated whether β-catenin could be a potential target to overcome endocrine resistance in breast cancer. Methods We established tamoxifen-resistant (TamR) cell line via long-term exposure of MCF-7 breast cancer cells to gradually increasing concentrations of tamoxifen. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of β-catenin was measured using luciferase activity assay. Results TamR cells showed a mesenchymal phenotype, and exhibited a relatively decreased expression of ER and increased expression of human epidermal growth factor receptor 2 and the epidermal growth factor receptor. We confirmed that the expression and transcriptional activity of β-catenin were increased in TamR cells compared with control cells. The expression and transcriptional activity of β-catenin were inhibited by β-catenin small-molecule inhibitor, ICG-001 or β-catenin siRNA. The viability of TamR cells, which showed no change after treatment with tamoxifen, was reduced by ICG-001 or β-catenin siRNA. The combination of ICG-001 and mTOR inhibitor, rapamycin, yielded an additive effect on the inhibition of viability in TamR cells. Conclusion These results suggest that β-catenin plays a role in tamoxifen-resistant breast cancer, and the inhibition of β-catenin may be a potential target in tamoxifen-resistant breast cancer. PMID:27196739

  3. 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.

  4. Protein translation inhibition by Stachybotrys chartarum conidia with and without the mycotoxin containing polysaccharide matrix.

    PubMed

    Karunasena, Enusha; Cooley, J Danny; Straus, Douglas; Straus, David C

    2004-07-01

    Recent studies have correlated the presence of Stachybotrys chartarum in structures with SBS. S. chartarum produces mycotoxins that are thought to produce some of the symptoms reported in sick-building syndrome (SBS). The conidia (spores) produced by Stachybotrys species are not commonly found in the air of buildings that have been found to contain significant interior growth of this organism. This could be due in part to the large size of the Stachybotrys spores, or the organism growing in hidden areas such as wall cavities. However, individuals in buildings with significant Stachybotrys growth frequently display symptoms that may be attributed to exposure to the organism's mycotoxins. In addition, Stachybotrys colonies produce a "slime" or polysaccharide (carbohydrate) matrix that coats the hyphae and the spores. The intent of this project was to determine whether the carbohydrate matrix and the mycotoxins embedded in it could be removed from the spores by repeated washings with either aqueous or organic solvents. The results demonstrated that the process of spore washing removed compounds that were toxic in a protein translation assay as compared to spores that were washed with an organic solution, however a correlation between carbohydrate removal during the washing process and the removal of mycotoxins from the spore surface was not observed. These data demonstrated that mycotoxins are not likely to be found exclusively in the carbohydrate matrix of the spores. Therefore, mycotoxin removal from the spore surface can occur without significant loss of polysaccharide. We also showed that toxic substances may be removed from the spore surface with an aqueous solution. These results suggest that satratoxins are soluble in aqueous solutions without being bound to water-soluble moieties, such as the carbohydrate slime matrix. PMID:15487326

  5. Angiotensin II increases fibronectin and collagen I through the β-catenin-dependent signaling in mouse collecting duct cells

    PubMed Central

    Cuevas, Catherina A.; Gonzalez, Alexis A.; Inestrosa, Nibaldo C.; Vio, Carlos P.

    2014-01-01

    The contribution of angiotensin II (ANG II) to renal and tubular fibrosis has been widely reported. Recent studies have shown that collecting duct cells can undergo mesenchymal transition suggesting that collecting duct cells are involved in interstitial fibrosis. The Wnt/β-catenin signaling pathway plays an essential role in development, organogenesis, and tissue homeostasis; however, the dysregulation of this pathway has been linked to fibrosis. In this study, we investigated whether AT1 receptor activation induces the expression of fibronectin and collagen I via the β-catenin pathway in mouse collecting duct cell line M-1. ANG II (10−7 M) treatment in M-1 cells increased mRNA, protein levels of fibronectin and collagen I, the β-catenin target genes (cyclin D1 and c-myc), and the myofibroblast phenotype. These effects were prevented by candesartan, an AT1 receptor blocker. Inhibition of the β-catenin degradation with pyrvinium pamoate (pyr; 10−9 M) prevented the ANG II-induced expression of fibronectin, collagen I, and β-catenin target genes. ANG II treatment promoted the accumulation of β-catenin protein in a time-dependent manner. Because phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits β-catenin degradation, we further evaluated the effects of ANG II and ANG II plus pyr on p-ser9-GSK-3β levels. ANG II-dependent upregulation of β-catenin protein levels was correlated with GSK-3β phosphorylation. These effects were prevented by pyr. Our data indicate that in M-1 collecting duct cells, the β-catenin pathway mediates the stimulation of fibronectin and collagen I in response to AT1 receptor activation. PMID:25411386

  6. Periostin is an extracellular matrix protein required for eruption of incisors in mice

    SciTech Connect

    Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira . E-mail: akudo@bio.titech.ac.jp

    2006-04-14

    A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin {sup -/-} mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin {sup -/-} mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.

  7. Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer.

    PubMed

    Gautron, J; Hincke, M T; Panheleux, M; Garcia-Ruiz, J M; Boldicke, T; Nys, Y

    2001-01-01

    The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80 kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

  8. Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro.

    PubMed

    Alibardi, Lorenzo; Polazzi, Elisabetta

    2012-04-01

    The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.

  9. Molecular Structure of a Peroxisomal Matrix Protein Transport Factor, Pex14p

    NASA Astrophysics Data System (ADS)

    Su, Jian-Rong; Takeda, Kazuki; Tamura, Shigehiko; Fujiki, Yukio; Miki, Kunio

    Peroxisome is an organelle in eukaryotic cells, which functions in various metabolisms such as β-oxidation of very long fatty acids. Peroxisomal matrix proteins synthesized in cytosol are imported into the peroxisome by a dynamic system consisting of over a dozen peroxins, Pex1p to Pex26p. Pex14p is a central component of the peroxisomal matrix protein import machinery. Until now, any structural information of Pex14p has not been elucidated at all. We describe here the crystal structure of the conserved domain of mammalian Pex14p at 1.8 Å resolution. A hydrophobic surface is composed of the conserved residues, of which two phenylalanine residues (Phe35 and Phe52) protrude to the solvent. Consequently, two putative binding pockets suitable for recognizing the helical WxxxF/Y motif of Pex5p are formed on the surface by the two phenylalanine residues accompanying with positively charged residues. Other structural studies for peroxins are also reviewed in this report.

  10. Growth factors and extracellular matrix proteins in interactions of cumulus-oocyte complex, spermatozoa and oviduct.

    PubMed

    Einspanier, R; Gabler, C; Bieser, B; Einspanier, A; Berisha, B; Kosmann, M; Wollenhaupt, K; Schams, D

    1999-01-01

    The expression and localization of selected growth factor systems and extracellular matrix (ECM) components that may influence oocyte maturation and fertilization within the mammalian oviduct are reported. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) systems could be detected by use of RT-PCR, RNase protection assay (RPA) and immunohistochemistry in bovine follicles, bovine cumulus-oocyte complexes (COC) and bovine and marmoset oviducts. Two different subtypes of the FGF receptor (FGFR-1 and -2) were identified in distinct cell types, indicating a functional difference. A complete epidermal growth factor (EGF) system was found in the porcine, but not in the bovine, oviduct. There were additional differences between bovine and primate oviducts: FGF-1/2 and FGFR were increased in the marmoset around ovulation, in contrast to an increase in FGF-1 in the cow. Immunohistochemistry revealed accumulation and storage of FGF and VEGF on the surface of the epithelium, possibly due to their binding property on heparanglycoproteins. Other ECM components, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1), were found to be modulated in the ovarian follicle, COC and oviduct during the cycle. An oviduct-mediated depletion of sperm surface proteins (BSP1-3) was discovered as well as a sperm-induced novel oviductal mRNA related to an anti-oxidant protein family. Associated systems of growth factors and ECM components can be suggested as paracrine or autocrine mediators during fertilization in a species-, cycle- and tissue-dependent manner.

  11. Crystal Structure of the Oligomeric Form of Lassa Virus Matrix Protein Z

    PubMed Central

    Hastie, Kathryn M.; Zandonatti, Michelle; Liu, Tong; Li, Sheng; Woods, Virgil L.

    2016-01-01

    ABSTRACT The arenavirus matrix protein Z is highly multifunctional and occurs in both monomeric and oligomeric forms. The crystal structure of a dodecamer of Z from Lassa virus, presented here, illustrates a ring-like structure with a highly basic center. Mutagenesis demonstrates that the dimeric interface within the dodecamer and a Lys-Trp-Lys triad at the center of the ring are important for oligomerization. This structure provides an additional template to explore the many functions of Z. IMPORTANCE The arenavirus Lassa virus causes hundreds of thousands of infections each year, many of which develop into fatal hemorrhagic fever. The arenavirus matrix protein Z is multifunctional, with at least four distinct roles. Z exists in both monomeric and oligomeric forms, each of which likely serves a specific function in the viral life cycle. Here we present the dodecameric form of Lassa virus Z and demonstrate that Z forms a “wreath” with a highly basic center. This structure and that of monomeric Z now provide a pair of critical templates by which the multiple roles of Z in the viral life cycle may be interpreted. PMID:26912609

  12. 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.

  13. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins.

    PubMed

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Khang, Gilson; Soker, Shay

    2013-02-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

  14. Augmented expression of urokinase plasminogen activator and extracellular matrix proteins associates with multiple myeloma progression.

    PubMed

    Khan, Rehan; Gupta, Nidhi; Kumar, Raman; Sharma, Manoj; Kumar, Lalit; Sharma, Alpana

    2014-06-01

    Multiple myeloma (MM) represents a B cell malignancy, characterized by a monoclonal proliferation of malignant plasma cells. Interactions between tumor cells and extracellular matrix (ECM) are of importance for tumor invasion and metastasis. Protein levels of urokinase plasminogen activator (uPA) and fibulin 1, nidogen and laminin in plasma and serum respectively and mRNA levels of these molecules in peripheral blood mononuclear cells were determined in 80 subjects by using ELISA and quantitative PCR and data was analyzed with severity of disease. Pearson correlation was determined to observe interrelationship between different molecules. A statistical significant increase for ECM proteins (laminin, nidogen and fibulin 1) and uPA at circulatory level as well as at mRNA level was observed compared to healthy controls. The levels of these molecules in serum might be utilized as a marker of active disease. Significant positive correlation of all ECM proteins with uPA was found and data also correlates with severity of disease. Strong association found between ECM proteins and uPA in this study supports that there might be interplay between these molecules which can be targeted. This study on these molecules may help to gain insight into processes of growth, spread, and clinical behavior of MM.

  15. Egg proteins as allergens and the effects of the food matrix and processing.

    PubMed

    Benedé, S; López-Expósito, I; Molina, E; López-Fandiño, R

    2015-03-01

    Hen eggs are an important and inexpensive source of high-quality proteins in the human diet. Egg, either as a whole or its constituents (egg yolk and white), is a key ingredient in many food products by virtue of its nutritional value and unique functional properties, such as emulsifying, foaming, and gelling. Nevertheless, egg is also known because of its allergenic potential and, in fact, it is the second most frequent source of allergic reactions, particularly in children. This review deals with the structural or functional properties of egg proteins that make them strong allergens. Their ability to sensitize and/or elicit allergic reactions is linked to their resistance to gastroduodenal digestion, which ultimately allows them to interact with the intestinal mucosa where absorption occurs. The factors that affect protein digestibility, whether increasing it, decreasing it, or inducing a different proteolysis pattern, and their influence on their capacity to induce or trigger an allergic reaction are discussed. Special attention is paid to the effect of the food matrix and the processing practices on the capacity of egg proteins to modulate the immune response.

  16. An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

    PubMed

    Schulenberg, Birte; Arnold, Brad; Patton, Wayne F

    2003-07-01

    Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.

  17. An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

    PubMed

    Schulenberg, Birte; Arnold, Brad; Patton, Wayne F

    2003-07-01

    Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively. PMID:12872220

  18. PAK4 Methylation by SETD6 Promotes the Activation of the Wnt/β-Catenin Pathway.

    PubMed

    Vershinin, Zlata; Feldman, Michal; Chen, Ayelet; Levy, Dan

    2016-03-25

    Lysine methylation of non-histone proteins has emerged as a key regulator of many cellular functions. Although less studied than other post-translational modifications such as phosphorylation and acetylation, the number of known methylated non-histone proteins is rapidly expanding. We have identified the p21-activated kinase 4 (PAK4) as a new substrate for methylation by the protein lysine methyltransferase SETD6. Our data demonstrate that SETD6 methylates PAK4 bothin vitroand at chromatin in cells. Interestingly, depletion of SETD6 in various cellular systems significantly hinders the activation of the Wnt/β-catenin target genes. PAK4 was recently shown to regulate β-catenin signaling, and we show that SETD6 is a key mediator of this pathway. In the presence of SETD6, the physical interaction between PAK4 and β-catenin is dramatically increased, leading to a significant increase in the transcription of β-catenin target genes. Taken together, our results uncover a new regulatory layer of the Wnt/β-catenin signaling cascade and provide new insight into SETD6 biology. PMID:26841865

  19. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  20. Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla.

    PubMed

    Rahman, M Azizur; Isa, Yeishin; Uehara, Tsuyoshi

    2005-03-01

    Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices

  1. The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

    PubMed Central

    de Oliveira Ferreira, Eliane; Teixeira, Felipe; Cordeiro, Fabiana; Lobo, Leandro Araujo; Rocha, Edson R.; Smith, Jeffrey C.; Domingues, Regina M C P

    2014-01-01

    Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis can not be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis. PMID:23850366

  2. Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows.

    PubMed

    Guo, Mengyao; Wang, Guoqing; Lv, Tingting; Song, Xiaojing; Wang, Tiancheng; Xie, Guanghong; Cao, Yongguo; Zhang, Naisheng; Cao, Rongfeng

    2014-03-15

    Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.

  3. Effect of pH, salt and chemical rinses on bacterial attachment to extracellular matrix proteins.

    PubMed

    Zulfakar, Siti Shahara; White, Jason D; Ross, Tom; Tamplin, Mark

    2013-06-01

    Microbial contamination of carcass surfaces occurs during slaughter and post-slaughter processing steps, therefore interventions are needed to enhance meat safety and quality. Although many studies have been done at the macro-level, little is known about specific processes that influence bacterial attachment to carcass surfaces, particularly the role of extracellular matrix (ECM) proteins. In the present study, the effect of pH and salt (NaCl, KCl and CaCl2) on attachment of Escherichia coli and Salmonella isolates to dominant ECM proteins: collagen I, fibronectin, collagen IV and laminin were assessed. Also, the effects of three chemical rinses commonly used in abattoirs (2% acetic acid, 2% lactic acid and 10% trisodium phosphate (TSP)) were tested. Within a pH range of 5-9, there was no significant effect on attachment to ECM proteins, whereas the effect of salt type and concentration varied depending on combination of strain and ECM protein. A concentration-dependant effect was observed with NaCl and KCl (0.1-0.85%) on attachment of E. coli M23Sr, but only to collagen I. One-tenth percent CaCl2 produced the highest level of attachment to ECM proteins for E. coli M23Sr and EC614. In contrast, higher concentrations of CaCl2 increased attachment of E. coli EC473 to collagen IV. Rinses containing TSP produced >95% reduction in attachment to all ECM proteins. These observations will assist in the design of targeted interventions to prevent or disrupt contamination of meat surfaces, thus improving meat safety and quality.

  4. Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

    PubMed Central

    Hill, C P; Worthylake, D; Bancroft, D P; Christensen, A M; Sundquist, W I

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus. Images Fig. 1 Fig. 2 Fig. 3 PMID:8610175

  5. Altered distribution of extracellular matrix proteins in the periodontal ligament of periostin-deficient mice.

    PubMed

    Tabata, Chihir