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Sample records for suppressor protein regulates

  1. Regulation of the Tumor Suppressor Protein PTEN by Phosphorylation

    National Research Council Canada - National Science Library

    Vasquez, Fancisca

    2001-01-01

    The purpose of the research project of this grant is to study the role of phosphorylation on the regulation of PTEN, a tumor suppressor localized on a chromosome region frequently deleted in various...

  2. Regulation of the Tumor Suppressor Protein PTEN by Phosphorylation

    National Research Council Canada - National Science Library

    Vazquez, Francisca

    2000-01-01

    The purpose of the research project of this grant is to study the role of phosphorylation on the regulation of PTEN, a tumor suppressor localized on a chromosome region frequently deleted in various...

  3. Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.

    Science.gov (United States)

    Kaur, Amanpreet; Westermarck, Jukka

    2016-12-15

    Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis by dephosphorylation of a variety of signaling proteins and acts as a tumor suppressor. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by highly complex mechanisms that are reviewed here. Importantly, recent studies have shown that PME-1 promotes oncogenic MAPK/ERK and AKT pathway activities in various cancer types. In human glioma, high PME-1 expression correlates with tumor progression and kinase inhibitor resistance. We discuss the emerging cancer-associated function of PME-1 and its potential clinical relevance. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  4. Regulation of IAP (Inhibitor of Apoptosis) Gene Expression by the p53 Tumor Suppressor Protein

    National Research Council Canada - National Science Library

    Murphy, Maureen

    2003-01-01

    The goal of the work proposed in this application, which has just completed Year 1, was to analyze the ability of the p53 tumor suppressor protein to repress the anti-apoptotic genes survivin and cIAP-2...

  5. 14-3-3 mediated regulation of the tumor suppressor protein, RASSF1A.

    Science.gov (United States)

    Ghazaleh, Haya Abu; Chow, Renfred S; Choo, Sheryl L; Pham, Diana; Olesen, Jamie D; Wong, Russell X; Onyskiw, Christina; Baksh, Shairaz

    2010-02-01

    Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFalpha) or TNFalpha related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFalpha receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3beta (GSK-3beta), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFalpha stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.

  6. The tumor suppressor, vitamin D3 up-regulated protein 1 (VDUP1), functions downstream of REPO during Drosophila gliogenesis.

    Science.gov (United States)

    Mandalaywala, Neil V; Chang, Solomon; Snyder, Randall G; Levendusky, Mark C; Voigt, Jeffrey M; Dearborn, Richard E

    2008-03-15

    The tumor suppressor, vitamin D(3) up-regulated protein 1 (VDUP1), regulates cell cycle progression by suppressing AP-1-dependent transcription. Loss of VDUP1 activity is associated with tumorigenesis but little is known about VDUP1 regulatory controls or developmental roles. Here we show that the Drosophila homolog of human VDUP1 (dVDUP1) is expressed throughout the nervous system at all stages of development, the first in vivo analysis of VDUP1 expression patterns in the brain. During neurogenesis dVDUP1 expression is transiently down-regulated coincident with neuroblast delamination. Subsequent to expression of the neuronal marker elav, dVDUP1 is up-regulated to varying degrees in developing neurons. In contrast, dVDUP1 expression is both robust and sustained during gliogenesis, and the cis-regulatory region of the dvdup1 gene contains consensus binding sites for the glial fate gene reversed polarity (repo). Expression of dVDUP1 in presumptive glia is lost in embryos deficient for the glial fate genes glial cells missing (gcm) and repo. Conversely, ectopic expression of gcm or repo was sufficient to induce dVDUP1 expression in the nervous system. Taken together, these data suggest a novel role for the dVDUP1 tumor suppressor during nervous system development as a regulatory target for REPO during gliogenesis.

  7. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway

    NARCIS (Netherlands)

    Arico, S.; Petiot, A.; Bauvy, C.; Dubbelhuis, P. F.; Meijer, A. J.; Codogno, P.; Ogier-Denis, E.

    2001-01-01

    The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic

  8. Hedgehog-dependent down-regulation of the tumor suppressor, vitamin D3 up-regulated protein 1 (VDUP1), precedes lamina development in Drosophila.

    Science.gov (United States)

    Chang, Solomon; Mandalaywala, Neil V; Snyder, Randall G; Levendusky, Mark C; Dearborn, Richard E

    2010-04-09

    The tumor suppressor vitamin D(3) up-regulated protein 1 (VDUP1) is expressed throughout the developing and mature Drosophila nervous system, but its regulatory pathways are not well understood. Within the developing Drosophila visual system, down-regulation of VDUP1 in lamina precursor cells (LPCs) coincided with the arrival of retinal axons into the lamina target field, suggesting VDUP1 regulation by an axonally transmitted signal. Hedgehog (Hh) is a signal well known to coordinate LPC proliferation and differentiation in response to retinal axon innervation, and analysis of orthologous dvdup1 promoters identified an evolutionarily conserved binding site for the Hh-dependent transcription factor cubitus interruptus (Ci). Hh-dependent regulation of VDUP1 in the developing lamina was confirmed in Hh loss-of-function backgrounds where VDUP1 expression was maintained in LPCs, inhibiting both cell proliferation and lamina neurogenesis. This putative coupling of VDUP1 to the Hh signaling pathway may provide novel insights into the mechanisms controlling brain growth and development. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  9. Suppressor of MEK null (SMEK)/protein phosphatase 4 catalytic subunit (PP4C) is a key regulator of hepatic gluconeogenesis.

    Science.gov (United States)

    Yoon, Young-Sil; Lee, Min-Woo; Ryu, Dongryeol; Kim, Jeong Ho; Ma, Hui; Seo, Woo-Young; Kim, Yo-Na; Kim, Su Sung; Lee, Chul Ho; Hunter, Tony; Choi, Cheol Soo; Montminy, Marc R; Koo, Seung-Hoi

    2010-10-12

    Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved in the regulation of hepatic glucose metabolism in mice. Expression of hepatic SMEK1/2 is up-regulated during fasting or in mouse models of insulin-resistant conditions in a Peroxisome Proliferator-Activated Receptor-gamma Coactivator 1α (PGC-1α)-dependent manner. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas depletion of the SMEK proteins reduces hyperglycemia and enhances CRTC2 phosphorylation; the effect is blunted by S171A CRTC2, which is refractory to salt-inducible kinase (SIK)-dependent inhibition. Taken together, we would propose that mammalian SMEK/PP4C proteins are involved in the regulation of hepatic glucose metabolism through dephosphorylation of CRTC2.

  10. Antiviral signaling protein MITA acts as a tumor suppressor in breast cancer by regulating NF-κB induced cell death.

    Science.gov (United States)

    Bhatelia, Khyati; Singh, Aru; Tomar, Dhanendra; Singh, Kritarth; Sripada, Lakshmi; Chagtoo, Megha; Prajapati, Paresh; Singh, Rochika; Godbole, Madan M; Singh, Rajesh

    2014-02-01

    Emerging evidences suggest that chronic inflammation is one of the major causes of tumorigenesis. The role of inflammation in regulation of breast cancer progression is not well established. Recently Mediator of IRF3 Activation (MITA) protein has been identified that regulates NF-κB and IFN pathways. Role of MITA in the context of inflammation and cancer progression has not been investigated. In the current report, we studied the role of MITA in the regulation of cross talk between cell death and inflammation in breast cancer cells. The expression of MITA was significantly lower on in estrogen receptor (ER) positive breast cancer cells than ER negative cells. Similarly, it was significantly down regulated in tumor tissue as compared to the normal tissue. The overexpression of MITA in MCF-7 and T47D decreases the cell proliferation and increases the cell death by activation of caspases. MITA positively regulates NF-κB transcription factor, which is essential for MITA induced cell death. The activation of NF-κB induces TNF-α production which further sensitizes MITA induced cell death by activation of death receptor pathway through capsase-8. MITA expression decreases the colony forming units and migration ability of MCF-7 cells. Thus, our finding suggests that MITA acts as a tumor suppressor which is down regulated during tumorigenesis providing survival advantage to tumor cell. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition

    International Nuclear Information System (INIS)

    Hackl, Christina; Stoeltzing, Oliver; Lang, Sven A; Moser, Christian; Mori, Akira; Fichtner-Feigl, Stefan; Hellerbrand, Claus; Dietmeier, Wolfgang; Schlitt, Hans J; Geissler, Edward K

    2010-01-01

    Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor

  12. Microbial Regulation of p53 Tumor Suppressor.

    Directory of Open Access Journals (Sweden)

    Alexander I Zaika

    2015-09-01

    Full Text Available p53 tumor suppressor has been identified as a protein interacting with the large T antigen produced by simian vacuolating virus 40 (SV40. Subsequent research on p53 inhibition by SV40 and other tumor viruses has not only helped to gain a better understanding of viral biology, but also shaped our knowledge of human tumorigenesis. Recent studies have found, however, that inhibition of p53 is not strictly in the realm of viruses. Some bacterial pathogens also actively inhibit p53 protein and induce its degradation, resulting in alteration of cellular stress responses. This phenomenon was initially characterized in gastric epithelial cells infected with Helicobacter pylori, a bacterial pathogen that commonly infects the human stomach and is strongly linked to gastric cancer. Besides H. pylori, a number of other bacterial species were recently discovered to inhibit p53. These findings provide novel insights into host-bacteria interactions and tumorigenesis associated with bacterial infections.

  13. Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.

    Directory of Open Access Journals (Sweden)

    Doug A Medvetz

    Full Text Available Birt-Hogg-Dube (BHD is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN, the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1 activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

  14. DMPD: Regulation of innate immunity by suppressor of cytokine signaling (SOCS)proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available oteins. Dalpke A, Heeg K, Bartz H, Baetz A. Immunobiology. 2008;213(3-4):225-35. Epub 2007 Nov 28. (.png) (....ignaling (SOCS)proteins. Authors Dalpke A, Heeg K, Bartz H, Baetz A. Publication Immunobiology. 2008;213(3-4

  15. BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1

    DEFF Research Database (Denmark)

    Carpenter, Brian; Hill, Kathryn J; Charalambous, Marika

    2004-01-01

    The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct e...

  16. Regulation of the tumor suppressor PTEN by natural anticancer compounds.

    Science.gov (United States)

    Kim, Do-Hee; Suh, Jinyoung; Surh, Young-Joon; Na, Hye-Kyung

    2017-08-01

    The tumor suppressor phosphatase and tensin homologue (PTEN) has phosphatase activity, with phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a product of phosphatidylinositol 3-kinase (PI3K), as one of the principal substrates. PTEN is a negative regulator of the Akt pathway, which plays a fundamental role in controlling cell growth, survival, and proliferation. Loss of PTEN function has been observed in many different types of cancer. Functional inactivation of PTEN as a consequence of germ-line mutations or promoter hypermethylation predisposes individuals to malignancies. PTEN undergoes posttranslational modifications, such as oxidation, acetylation, phosphorylation, SUMOylation, and ubiquitination, which influence its catalytic activity, interactions with other proteins, and subcellular localization. Cellular redox status is crucial for posttranslational modification of PTEN and its functional consequences. Oxidative stress and inflammation are major causes of loss of PTEN function. Pharmacologic or nutritional restoration of PTEN function is considered a reliable strategy in the management of PTEN-defective cancer. In this review, we highlight natural compounds, such as curcumin, indol-3 carbinol, and omega-3 fatty acids, that have the potential to restore or potentiate PTEN expression/activity, thereby suppressing cancer cell proliferation, survival, and resistance to chemotherapeutic agents. © 2017 New York Academy of Sciences.

  17. Identification of a maize chlorotic dwarf virus silencing suppressor protein.

    Science.gov (United States)

    Stewart, Lucy R; Jarugula, Sridhar; Zhao, Yujing; Qu, Feng; Marty, DeeMarie

    2017-04-01

    Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389kDa proteolytically processed polyprotein. We show that the N-terminal 78kDa polyprotein (R78) of MCDV acts as a suppressor of RNA silencing in a well-established assay system. We further demonstrate that R78 is cleaved by the viral 3C-like protease into 51 and 27kDa proteins (p51 and p27), and that p51 is responsible for silencing suppressor activity. Silencing suppressor activity of R78 is conserved in three divergent MCDV strains (MCDV-Severe, MCDV-M1, and MCDV-Tennessee), as well as the waikavirus Bellflower vein chlorosis virus, but was not detected for orthologous protein of Rice tungro spherical virus (RTSV-A) or the similarly-positioned protein from the sequivirus Parsnip yellow fleck virus (PYFV). This is the first identification of a virus suppressor of RNA silencing encoded by a waikavirus. Copyright © 2017. Published by Elsevier Inc.

  18. The Retinoblastoma Tumor Suppressor Regulates a Xenobiotic Detoxification Pathway

    Science.gov (United States)

    Sáenz Robles, Maria Teresa; Case, Ashley; Chong, Jean-Leon; Leone, Gustavo; Pipas, James M.

    2011-01-01

    The retinoblastoma tumor suppressor (pRb) regulates cell cycle entry, progression and exit by controlling the activity of the E2F-family of transcription factors. During cell cycle exit pRb acts as a transcriptional repressor by associating with E2F proteins and thereby inhibiting their ability to stimulate the expression of genes required for S phase. Indeed, many tumors harbor mutations in the RB gene and the pRb-E2F pathway is compromised in nearly all types of cancers. In this report we show that both pRb and its interacting partners, the transcriptional factors E2F1-2-3, act as positive modulators of detoxification pathways important for metabolizing and clearing xenobiotics—such as toxins and drugs—from the body. Using a combination of conventional molecular biology techniques and microarray analysis of specific cell populations, we have analyzed the detoxification pathway in murine samples in the presence or absence of pRb and/or E2F1-2-3. In this report, we show that both pRb and E2F1-2-3 act as positive modulators of detoxification pathways in mice, challenging the conventional view of E2F1-2-3 as transcriptional repressors negatively regulated by pRb. These results suggest that mutations altering the pRb-E2F axis may have consequences beyond loss of cell cycle control by altering the ability of tissues to remove toxins and to properly metabolize anticancer drugs, and might help to understand the formation and progression rates of different types of cancer, as well as to better design appropriate therapies based on the particular genetic composition of the tumors. PMID:22022495

  19. Modulator of apoptosis 1 (MOAP-1) is a tumor suppressor protein linked to the RASSF1A protein.

    Science.gov (United States)

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R B; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C; Mackey, John; Baksh, Shairaz

    2015-10-02

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

    Science.gov (United States)

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

    2015-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

  1. Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

    Directory of Open Access Journals (Sweden)

    Ziad J. Sahab, Michael D. Hall, Lihua Zhang, Amrita K. Cheema, Stephen W. Byers

    2010-01-01

    Full Text Available Retinoic Acid Receptor Responder (RARRES1 initially identified as a novel retinoic acid receptor regulated gene in the skin is a putative tumor suppressor of unknown function. RARRES1 was knocked down in immortalized human prostatic epithelial cell line PWR-1E cells and differential protein expression was identified using differential in-gel electrophoresis (DIGE followed by matrix-assisted laser desorption ionization (MALDI mass spectrometry and western Blot analysis excluding highly abundant proteins routinely identified in almost all proteomics projects. Knock-down of RARRES1: 1- down-regulates PP2A, an enzyme involved in the negative regulation of the growth hormone-stimulated signal transduction pathways; 2- down-regulates Valosin-containing protein causing impaired autophagy; 3- up-regulates the tumor suppressor disks large 2; 4- up-regulates Ankrd26 that belongs to the POTE family of genes that are highly expressed in cancer patients with poor outcome; and 5- down-regulates EB1, a protein that is involved in spindle dynamics and chromosome alignment during mitosis.

  2. The tumor suppressor phosphatase and tensin homolog protein (PTEN) is negatively regulated by NF-κb p50 homodimers and involves histone 3 methylation/deacetylation in UROtsa cells chronically exposed to monomethylarsonous acid.

    Science.gov (United States)

    Oliva-González, C; Uresti-Rivera, E E; Galicia-Cruz, O G; Jasso-Robles, F I; Gandolfi, A J; Escudero-Lourdes, C

    2017-10-05

    UROtsa cells have been accepted as a model to study carcinogenicity mechanisms of arsenic-associated human bladder cancer. In vitro continuous exposure to monomethylarsonous acid (MMA III ), leads UROtsa cells to commit to malignant transformation. In this process, NF-κβ-associated inflammatory response seems to play an important role since this transcription factor activates some minutes after cells are exposed in vitro to MMA III and keeps activated during the cellular malignant transformation. It is known that a slight decrease in the protein phosphatase and tensin homologue (PTEN) gene expression is enough for some cells to become malignantly transformed. Interestingly, this tumor suppressor has been proven to be negatively regulated by NF-κβ through binding to its gene promoter. Based on these observations we propose that NF-κβ may be involved in arsenic associated carcinogenesis through the negative regulation of PTEN gene expression. Changes in PTEN expression and the binding of p50 NF-κβ subunit to PTEN promoter were evaluated in UROtsa cells exposed for 4, 12, 20, or 24 wk to 50nM MMA III . Results showed that MMA III induced a significant decrease in PTEN expression around 20 wk exposure to MMA III ,which correlated with increased binding of p50 subunit to the PTEN promoter. Consistent with these results, ChIP assays also showed a significant decrease in H3 acetylation (H3ac) but an increase in the repression marks H3k9me3 and H327me3 in PTEN promoter when compared with not treated cells. These results suggest that the activation of NF-κβ by MMA III may participate in UROtsa cells malignant transformation through the negative regulation of PTEN expression involving p50 homodimers-mediated chromatin remodeling around the PTEN promoter. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Stephen R. Armstrong

    2016-12-01

    Full Text Available The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

  4. Dissecting functions of the retinoblastoma tumor suppressor and the related pocket proteins by integrating genetic, cell biology, and electrophoretic techniques

    DEFF Research Database (Denmark)

    Hansen, Klaus; Lukas, J; Holm, K

    1999-01-01

    The members of the 'pocket protein' family, comprising the retinoblastoma tumor suppressor (pRB) and its relatives, p107 and p130, negatively regulate cell proliferation and modulate fundamental biological processes including embryonic development, differentiation, homeostatic tissue renewal......, and defense against cancer. The large, multidomain pocket proteins act by binding a plethora of cell fate-determining and growth-stimulatory proteins, the most prominent of which are the E2F/DP transcription factors. These protein-protein interactions are in turn regulated by carefully orchestrated...

  5. PML tumor suppressor protein is required for HCV production

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, Misao [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Research Fellow of the Japan Society for the Promotion of Science (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Ariumi, Yasuo, E-mail: ariumi@kumamoto-u.ac.jp [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Center for AIDS Research, Kumamoto University, Kumamoto 860-0811 (Japan); Hijikata, Makoto [Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Ikeda, Masanori; Dansako, Hiromichi [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan); Wakita, Takaji [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Shimotohno, Kunitada [Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Chiba 272-8516 (Japan); Kato, Nobuyuki [Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558 (Japan)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  6. Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias.

    Science.gov (United States)

    Fang, Hongbo; Liu, Jing; Guo, Dan; Liu, Peixiang; Zhao, Yongliang

    2014-01-01

    Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types. The hypermethylation of the TGFBI promoter, as one of the main regulatory mechanisms, is associated with TGFBI silencing. In this study, we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias. Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia cell lines and clinical samples. Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients, bisulfite-converted, and analyzed by the MSP method. Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested. Furthermore, a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples. The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia, which provides a useful diagnostic marker for clinical management of human leukemias.

  7. Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Neal Corey L

    2012-08-01

    Full Text Available Abstract Background Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT. We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells. Methods Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP assays. Results Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells. Conclusions Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

  8. Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

    OpenAIRE

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John

    2015-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databas...

  9. ATP and MO25? Regulate the Conformational State of the STRAD? Pseudokinase and Activation of the LKB1 Tumour Suppressor

    OpenAIRE

    Zeqiraj, Elton; Filippi, Beatrice Maria; Goldie, Simon; Navratilova, Iva; Boudeau, J?r?me; Deak, Maria; Alessi, Dario R.; van Aalten, Daan M. F.

    2009-01-01

    Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the me...

  10. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  11. The Tumor Suppressor Protein TEP1/PTEN/MMAC1 and Human Breast Cancer

    National Research Council Canada - National Science Library

    Sun, Hong

    2002-01-01

    PTEN is an important tumor suppressor. Both inherited mutations and somatic mutations in the PTEN gene have been frequently found in a variety of human cancers, including the breast cancer, PTEN protein has been shown to possess...

  12. PDZ-containing 1 acts as a suppressor of pancreatic cancer by regulating PTEN phosphorylation.

    Science.gov (United States)

    Ma, Qiang; Wu, Xiuxiu; Wu, Jing; Wu, Huanwen; Xiao, Ying; Wang, Lili; Liang, Zhiyong; Liu, Tonghua

    2017-09-22

    Phosphorylation is a recently established cause of phosphatase and tensin homolog (PTEN) gene inactivation, which leads to defect tumour-suppressor function. In pancreatic cancer, this phenomenon has not been reported. Based on database and clinical sample analyses, we found that PTEN phosphorylation occurs in pancreatic ductal adenocarcinoma patient tissues and cell lines, and we aimed to find a method for dephosphorylation. PDZ-containing 1 (PDZK1), a tumour-associated protein that shares its PDZ-binding sequence with the carboxyl-terminal domain of PTEN, was significantly down-regulated in pancreatic cancer as compared to adjacent non-tumour tissues. In vitro , PDZK1 overexpression reversed the proliferation and migration abilities of pancreatic cancer cells and led to significantly decreased PTEN phosphorylation and AKT phosphorylation by interacting with wild-type PTEN. In addition, a transcription factor-activation assay supported that PDZK1 overexpression enhanced the anti-oncogene function of PTEN by regulating the activities of its downstream transcription factors, including p53, NF-κB, and FOXO1. In vivo , nude mice stably over-expressing PDZK1 had lower tumour weights and volumes and showed significantly down-regulated PTEN phosphorylation in xenograft tumour tissues as compared to the control group. Moreover, low PDZK1 expression strongly correlated with advanced stage and poor prognosis of patients with pancreatic ductal adenocarcinoma. In conclusion, our study elucidated the tumour-suppressor role of PDZK1 in pancreatic cancer through down-regulating PTEN phosphorylation, and established PDZK1 as a potential novel prognostic marker for pancreatic cancer.

  13. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  14. Methylation of the tumor suppressor protein, BRCA1, influences its transcriptional cofactor function.

    Directory of Open Access Journals (Sweden)

    Irene Guendel

    Full Text Available BACKGROUND: Approximately half of hereditary breast cancers have mutations in either BRCA1 or BRCA2. BRCA1 is a multifaceted tumor suppressor protein that has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. This multifunctional nature of BRCA1 is achieved by exerting its many effects through modulation of transcription. Many cellular events are dictated by covalent modification of proteins, an important mechanism in regulating protein and genome function; of which protein methylation is an important posttranslational modification with activating or repressive effects. METHODS/PRINCIPAL FINDINGS: Here we demonstrate for the first time that BRCA1 is methylated both in breast cancer cell lines and breast cancer tumor samples at arginine and lysine residues through immunoprecipitation and western blot analysis. Arginine methylation by PRMT1 was observed in vitro and the region of BRCA1 504-802 shown to be highly methylated. PRMT1 was detected in complex with BRCA1 504-802 through in vitro binding assays and co-immunoprecipitated with BRCA1. Inhibition of methylation resulted in decreased BRCA1 methylation and alteration of BRCA1 binding to promoters in vivo as shown through chromatin immunoprecipitation assays. Knockdown of PRMT1 also resulted in increased BRCA1 binding to particular promoters in vivo. Finally, following methylation inhibition, Sp1 was found to preferentially associate with hypo-methylated BRCA1 and STAT1 was found to preferentially associate with hyper-methylated BRCA1. CONCLUSIONS/SIGNIFICANCE: These results suggest that methylation may influence either the ability of BRCA1 to bind to specific promoters or protein-protein interactions which alters the recruitment of BRCA1 to these promoters. Thus, given the importance of BRCA1 to genomic stability, methylation of BRCA1 may ultimately affect the tumor suppressor ability of BRCA1.

  15. Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis

    Science.gov (United States)

    Boutté, Angela M.; Friedman, David B.; Bogyo, Matthew; Min, Yongfen; Yang, Li; Lin, P. Charles

    2011-01-01

    Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ∼40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.—Boutté, A. M., Friedman, D. B., Bogyo, M., Min, Y., Yang, L., Lin, P. C. Identification of a myeloid-derived suppressor cell cystatin-like protein that inhibits metastasis. PMID:21518852

  16. Tumor Suppressors and Cell-Cycle Proteins in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Alfonso Baldi

    2011-01-01

    Full Text Available The cell cycle is the cascade of events that allows a growing cell to duplicate all its components and split into two daughter cells. Cell cycle progression is mediated by the activation of a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs. CDKs are also regulated by related proteins called cdk inhibitors grouped into two families: the INK4 inhibitors (p16, p15, p19, and p18 and the Cip/Kip inhibitors (p21, p27, and p53. Several studies report the importance of cell-cycle proteins in the pathogenesis and the prognosis of lung cancer. This paper will review the most recent data from the literature about the regulation of cell cycle. Finally, based essentially on the data generated in our laboratory, the expression, the diagnostic, and prognostic significance of cell-cycle molecules in lung cancer will be examined.

  17. Identification of a maize chlorotic dwarf virus silencing suppressor protein

    Science.gov (United States)

    Maize chlorotic dwarf virus (MCDV), a member of the genus Waikavirus, family Secoviridae, has a 11784 nt (+)ssRNA genome that encodes a 389 kDa proteolytically processed polyprotein. We show that an N-terminal 78kDa polyprotein (R78) has silencing suppressor activity, that it is cleaved by the viral...

  18. Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

    Science.gov (United States)

    Kotake, Yojiro; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Niida, Hiroyuki; Naemura, Madoka; Kitagawa, Masatoshi

    2016-04-01

    P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    Directory of Open Access Journals (Sweden)

    Mamgain Hitesh

    2009-10-01

    Full Text Available Abstract Background Imaging tools such as scanning electron microscope (SEM and atomic force microscope (AFM can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK 293, human breast cancer (MCF-7 and mouse melanoma (B16F1 cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM and scanning electron microscopy (SEM. The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor

  20. Genistein up-regulates tumor suppressor microRNA-574-3p in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Takeshi Chiyomaru

    Full Text Available Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs. In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1 and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as 'Pathways in cancer', 'Jak-STAT signaling pathway', and 'Wnt signaling pathway'. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3' UTR of several target genes (such as RAC1, EGFR and EP300 that are components of 'Pathways in cancer'. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa.

  1. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  2. Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

    2003-06-11

    Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

  3. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  4. Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    2017-05-01

    Full Text Available Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx system. Here, the role of tert-butyl hydroperoxide (t-BHP in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.

  5. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    International Nuclear Information System (INIS)

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  6. Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide.

    Science.gov (United States)

    Zhang, Ying; Han, Seong-Jeong; Park, Iha; Kim, Inyoung; Chay, Kee-Oh; Kim, Seok Mo; Jang, Dong Il; Lee, Tae-Hoon; Lee, Seung-Rock

    2017-05-10

    Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert -butyl hydroperoxide ( t -BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t -BHP led to oxidation of recombinant PTEN. In contrast to H₂O₂, PTEN oxidation by t -BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t -BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t -BHP in the promotion of tumorigenesis.

  7. Intragenic suppressor of Osiaa23 revealed a conserved tryptophan residue crucial for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Jun Ni

    Full Text Available The Auxin/Indole-3-Acetic Acid (Aux/IAA and Auxin Response Factor (ARF are two important families that play key roles in auxin signal transduction. Both of the families contain a similar carboxyl-terminal domain (Domain III/IV that facilitates interactions between these two families. In spite of the importance of protein-protein interactions among these transcription factors, the mechanisms involved in these interactions are largely unknown. In this study, we isolated six intragenic suppressors of an auxin insensitive mutant, Osiaa23. Among these suppressors, Osiaa23-R5 successfully rescued all the defects of the mutant. Sequence analysis revealed that an amino acid substitution occurred in the Tryptophan (W residue in Domain IV of Osiaa23. Yeast two-hybrid experiments showed that the mutation in Domain IV prevents the protein-protein interactions between Osiaa23 and OsARFs. Phylogenetic analysis revealed that the W residue is conserved in both OsIAAs and OsARFs. Next, we performed site-specific amino acid substitutions within Domain IV of OsARFs, and the conserved W in Domain IV was exchanged by Serine (S. The mutated OsARF(WSs can be released from the inhibition of Osiaa23 and maintain the transcriptional activities. Expression of OsARF(WSs in Osiaa23 mutant rescued different defects of the mutant. Our results suggest a previously unknown importance of Domain IV in both families and provide an indirect way to investigate functions of OsARFs.

  8. Influence of anticancer drugs on interactions of tumor suppressor protein p53 with DNA

    Czech Academy of Sciences Publication Activity Database

    Pivoňková, Hana; Němcová, Kateřina; Brázdová, Marie; Kašpárková, Jana; Brabec, Viktor; Fojta, Miroslav

    2005-01-01

    Roč. 272, Suppl. 1 (2005), s. 562 ISSN 1474-3833. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] R&D Projects: GA MZd(CZ) NC7574 Institutional research plan: CEZ:AV0Z50040507 Keywords : tumour suppressor protein p53 * anticancer drugs * interaction with DNA Subject RIV: BO - Biophysics

  9. Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Song, Zuohe; Saghafi, Negin; Gokhale, Vijay; Brabant, Marc; Meuillet, Emmanuelle J.

    2007-01-01

    Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies

  10. Saponin Inhibits Hepatitis C Virus Propagation by Up-regulating Suppressor of Cytokine Signaling 2

    Science.gov (United States)

    Kang, Sang-Min; Min, Saehong; Son, Kidong; Lee, Han Sol; Park, Eun Mee; Ngo, Huong T. T.; Tran, Huong T. L.; Lim, Yun-Sook; Hwang, Soon B.

    2012-01-01

    Saponins are a group of naturally occurring plant glycosides which possess a wide range of pharmacological properties, including anti-tumorigenic and antiviral activities. To investigate whether saponin has anti-hepatitis C virus (HCV) activity, we examined the effect of saponin on HCV replication. HCV replication was efficiently inhibited at a concentration of 10 µg/ml of saponin in cell culture grown HCV (HCVcc)-infected cells. Inhibitory effect of saponin on HCV replication was verified by quantitative real-time PCR, reporter assay, and immunoblot analysis. In addition, saponin potentiated IFN-α-induced anti-HCV activity. Moreover, saponin exerted antiviral activity even in IFN-α resistant mutant HCVcc-infected cells. To investigate how cellular genes were regulated by saponin, we performed microarray analysis using HCVcc-infected cells. We demonstrated that suppressor of cytokine signaling 2 (SOCS2) protein level was distinctively increased by saponin, which in turn resulted in inhibition of HCV replication. We further showed that silencing of SOCS2 resurrected HCV replication and overexpression of SOCS2 suppressed HCV replication. These data imply that saponin inhibits HCV replication via SOCS2 signaling pathway. These findings suggest that saponin may be a potent therapeutic agent for HCV patients. PMID:22745742

  11. Electrochemical sensing of tumor suppressor protein p53-deoxyribonucleic acid complex stability at an electrified interface

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Černocká, Hana; Ostatná, Veronika; Navrátilová, Lucie; Brázdová, Marie

    2014-01-01

    Roč. 828, MAY2014 (2014), s. 1-8 ISSN 0003-2670 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GA13-00956S; GA ČR(CZ) GA13-36108S Institutional support: RVO:68081707 Keywords : Deoxyribonucleic acid-protein binding * Tumor suppressor protein p53 * Electrochemical sensing Subject RIV: BO - Biophysics Impact factor: 4.513, year: 2014

  12. Estrogen receptor β regulates the tumoral suppressor PTEN to modulate pituitary cell growth.

    Science.gov (United States)

    Perez, Pablo A; Petiti, Juan P; Picech, Florencia; Guido, Carolina B; dV Sosa, Liliana; Grondona, Ezequiel; Mukdsi, Jorge H; De Paul, Ana L; Torres, Alicia I; Gutierrez, Silvina

    2018-02-01

    In this study, we focused on ERβ regulation in the adenohypophysis under different estrogenic milieu, by analyzing whether ER modulates the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression and its subcellular localization on anterior pituitary glands from Wistar rats and GH3 lactosomatotroph cells that over-expressed ERβ. ERβ was regulated in a cyclic manner, and underwent dynamic changes throughout the estrous cycle, with decreased ERβ+ cells in estrus and under E2 treatment, but increased in ovariectomized rats. In addition, the ERα/β ratio increased in estrus and under E2 stimulation, but decreased in ovariectomized rats. Double immunofluorescence revealed that lactotroph and somatotroph ERβ+ were significantly decreased in estrus. Also, variations in the PTEN expression was observed, which was diminished with high E2 conditions but augmented with low E2 milieu. The subcellular localization of this phosphatase was cell cycle-dependent, with remarkable changes in the immunostaining pattern: nuclear in arrested pituitary cells but cytoplasmic in stimulated cells, and responding differently to ER agonists, with only DPN being able to increase PTEN expression and retaining it in the nucleus. Finally, ERβ over-expression increased PTEN with a noticeable subcellular redistribution, and with a significant nuclear signal increase in correlation with an increase of cells in G0/G1 phase. These results showed that E2 is able to inhibit ERβ expression and suggests that the tumoral suppressor PTEN might be one of the signaling proteins by which E2, through ERβ, acts to modulate pituitary cell proliferation, thereby adapting endocrine populations in relation with hormonal necessities. © 2017 Wiley Periodicals, Inc.

  13. Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

    Science.gov (United States)

    Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

    2017-10-15

    Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers. IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and

  14. Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene

    Science.gov (United States)

    Ifere, Godwin O.; Equan, Anita; Gordon, Kereen; Nagappan, Peri; Igietseme, Joseph U.; Ananaba, Godwin A.

    2010-01-01

    Background The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream regulated gene1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. Methods PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72 h, followed by trypan blue dye exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. Results Physiological doses (16 µM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol enrichment promoted cell growth (Pphytosterols significantly induced growth-suppression (Pphytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1(cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. Conclusions Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor-suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling

  15. Negative Regulation of the Stability and Tumor Suppressor Function of Fbw7 by the Pin1 Prolyl Isomerase

    Science.gov (United States)

    Min, Sang-Hyun; Lau, Alan W.; Lee, Tae Ho; Inuzuka, Hiroyuki; Wei, Shuo; Huang, Pengyu; Shaik, Shavali; Lee, Daniel Yenhong; Finn, Greg; Balastik, Martin; Chen, Chun-Hau; Luo, Manli; Tron, Adriana E.; DeCaprio, James A.; Zhou, Xiao Zhen; Wei, Wenyi; Lu, Kun Ping

    2012-01-01

    SUMMARY Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy. PMID:22608923

  16. Immunopurification of the suppressor tRNA dependent rabbit β-globin readthrough protein

    International Nuclear Information System (INIS)

    Hatfield, D.; Thorgeirsson, S.S.; Copeland, T.D.; Oroszlan, S.; Bustin, M.

    1988-01-01

    In mammalian cells, the rabbit β-globin readthrough protein is the only known example of a naturally occurring readthrough protein which does not involve a viral system. To provide an efficient means for its isolation, detection, and study, the authors elicited specific antibodies against this unique protein. The 22 amino acid peptide corresponding to the readthrough portion of this protein was synthesized, coupled to keyhole limpet hemocyanin, and injected into sheep. Specific antibodies to the peptide were produced as demonstrated by the enzyme-linked immunosorbent assay technique and by immunoblotting. The antibodies did not react with globin. The rabbit β-globin readthrough protein was separated from globin and other reticulocyte proteins by polyacrylamide gel electrophoresis and visualized by silver staining or by labeling with [ 35 S] methionine. Incorporation of [ 35 S] methionine into the readthrough protein was significantly enhanced upon addition of an opal suppressor tRNA to reticulocyte lysates. Immunoblotting revealed that the readthrough protein also occurs in lysates without added suppressor tRNA. The antibodies were purified on an affi-gel column which had been coupled with the peptide antigen. The readthrough protein was then purified from reticulocytes by immunoaffinity chromatography and by high-performance liquid chromatography. The results provide conclusive evidence that the β-globin readthrough protein is naturally occurring in rabbit reticulocytes

  17. Clinical applications of detecting dysfunctional p53 tumor suppressor protein

    NARCIS (Netherlands)

    Baas, I. O.; Hruban, R. H.; Offerhaus, G. J.

    1999-01-01

    The p53 gene encodes for a protein, p53, which plays a critical role in controlling the cell cycle, in DNA repair and in programmed cell death (apoptosis). p53 is one of the most frequently mutated genes in human neoplasms and a variety of techniques have been developed to detect these mutations.

  18. Neurofibromatosis type 2 tumor suppressor protein, NF2, induces proteasome-mediated degradation of JC virus T-antigen in human glioblastoma.

    Directory of Open Access Journals (Sweden)

    Sarah Beltrami

    Full Text Available Neurofibromatosis type 2 protein (NF2 has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV tumor antigen (T-antigen as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.

  19. Regulation of transcription by the retinoblastoma protein.

    Science.gov (United States)

    Horowitz, J M

    1993-02-01

    The product of the retinoblastoma gene (RB1) is believed to function as a negative regulator of cell growth. Recent experimental results suggest that RB1 may exert its growth-suppressing activity by regulating the transcription of a variety of growth-related genes, including FOS, MYC, and TGFBI. A series of biochemical and molecular analyses suggest that RB1 indirectly affects gene expression via cell-cycle-regulated interactions with transcription factors, such as E2F and SPI. Determination of the mechanisms regulating such protein-protein interactions and the identification of additional targets of RB1 function will provide vital insights into the role of this tumor-suppressor gene in mammalian cell proliferation.

  20. Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells

    Directory of Open Access Journals (Sweden)

    Maryam Zangi

    2016-07-01

    Full Text Available Background: The potential of plants, as a safe and eukaryotic system, is considered in the production of recombinant therapeutic human protein today; but the expression level of heterologous proteins is limited by the post-transcriptional gene silencing (PTGS response in this new technology. The use of viral suppressors of gene silencing can prevent PTGS and improve transient expression level of foreign proteins. In this study, we investigated the effect of p19 silencing suppressor on recombinant human nerve growth factor expression in Nicotiana benthamiana. Materials and Methods: The p19 coding region was inserted in the pCAMBIA using NcoI and BstEII recognition sites. Also, the cloned synthesized recombinant human NGF (rhNGF fragment was cloned directly into PVX vector by ClaI and SalI restriction enzymes. The co-agroinfiltration of rhNGF with p19 viral suppressor of gene silencing was evaluated by dot-blot and SDS-PAGE. The amount of expressed rhNGF protein was calculated by AlphaEaseFC software. Results: Co-agroinfiltration of hNGF with P19 suppressor showed about forty-fold increase (8% total soluble protein (TSP when compared to the absence of P19 suppressor (0.2%TSP. Conclusion: The results presented here confirmed that the use of P19 gene silencing suppressor derived from tomato bushy stunt virus (TBSV could efficiently increase the transient expression of recombinant proteins in Nicotiana benthamiana manifold.

  1. The tumor suppressors p53, p63, and p73 are regulators of microRNA processing complex.

    Directory of Open Access Journals (Sweden)

    Lakshmanane Boominathan

    2010-05-01

    Full Text Available The tumor suppressors p53, p73, and p63 are known to function as transcription factors. They promote either growth arrest or apoptosis, depending upon the DNA damage. A number of microRNAs (miRNAs have been shown to function as transcriptional targets of p53 and they appear to aid p53 in promoting growth arrest and apoptosis. However, the question of p53/p63/p73 regulating the miRNA processing complex has not been addressed in depth so far. Comparative/computational genomic analysis was performed using Target scan, Mami, and Diana software to identify miRNAs that regulate the miRNA processing complex. Here, I present evidence for the first time that the tumor suppressors p53, p63, and p73 function as both positive and negative regulators of the miRNA processing components. Curated p53-dependent miRNA expression data was used to identify p53-miRs that target the components of the miRNA-processing complex. This analysis suggests that most of the components (mRNAs' 3'UTR of the miRNA processing complex are targeted by p53-miRs. Remarkably, this data revealed the conserved nature of p53-miRs in targeting a number of components of the miRNA processing complex. p53/p73/p63 appears to regulate the major components of the miRNA processing, such as Drosha-DGCR8, Dicer-TRBP2, and Argonaute proteins. In particular, p53/p73/p63 appears to regulate the processing of miRNAs, such as let-7, miR-200c, miR-143, miR-107, miR-16, miR-145, miR-134, miR-449a, miR-503, and miR-21. Interestingly, there seems to be a phenotypic similarity between p63(-/- and dicer(-/- mice, suggesting that p63 and dicer could regulate each other. In addition, p63, p73, and the DGCR8 proteins contain a conserved interaction domain. Further, promoters of a number of components of the miRNA processing machinery, including dicer and P2P-R, contain p53-REs, suggesting that they could be direct transcriptional targets of p63/p73/p53. Together, this study provides mechanistic insights into

  2. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    For example, the structural changes that allowed for allosteric regulation of haemoglobin were re- vealed through structural elucidation of the protein in free and oxygen-bound forms by X-ray crystallography. Following this,. X-ray crystallography has been utilized to study a variety of al- losteric proteins including ATCase. 2.

  3. Analysis of geminivirus AL2 and L2 proteins reveals a novel AL2 silencing suppressor activity.

    Science.gov (United States)

    Jackel, Jamie N; Buchmann, R Cody; Singhal, Udit; Bisaro, David M

    2015-03-01

    Both posttranscriptional and transcriptional gene silencing (PTGS and TGS, respectively) participate in defense against the DNA-containing geminiviruses. As a countermeasure, members of the genus Begomovirus (e.g., Cabbage leaf curl virus) encode an AL2 protein that is both a transcriptional activator and a silencing suppressor. The related L2 protein of Beet curly top virus (genus Curtovirus) lacks transcription activation activity. Previous studies showed that both AL2 and L2 suppress silencing by a mechanism that correlates with adenosine kinase (ADK) inhibition, while AL2 in addition activates transcription of cellular genes that negatively regulate silencing pathways. The goal of this study was to clarify the general means by which these viral proteins inhibit various aspects of silencing. We confirmed that AL2 inhibits systemic silencing spread by a mechanism that requires transcription activation activity. Surprisingly, we also found that reversal of PTGS and TGS by ADK inactivation depended on whether experiments were conducted in vegetative or reproductive Nicotiana benthamiana plants (i.e., before or after the vegetative-to-reproductive transition). While AL2 was able to reverse silencing in both vegetative and reproductive plants, L2 and ADK inhibition were effective only in vegetative plants. This suggests that silencing maintenance mechanisms can change during development or in response to stress. Remarkably, we also observed that AL2 lacking its transcription activation domain could reverse TGS in reproductive plants, revealing a third, previously unsuspected AL2 suppression mechanism that depends on neither ADK inactivation nor transcription activation. RNA silencing in plants is a multivalent antiviral defense, and viruses respond by elaborating multiple and sometimes multifunctional proteins that inhibit various aspects of silencing. The studies described here add an additional layer of complexity to this interplay. By examining geminivirus AL2 and

  4. Regulation of cellular transport by klotho protein.

    Science.gov (United States)

    Sopjani, Mentor; Rinnerthaler, Mark; Almilaji, Ahmad; Ahmeti, Salih; Dermaku-Sopjani, Miribane

    2014-01-01

    The antiaging protein of Klotho is a transmembrane protein mainly expressed in the kidney, parathyroid glands and choroid plexus of the brain. The Klotho protein exists in two forms, a full-length membrane form and a soluble secreted form. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it acts as β-glucuronidase and a hormone. Soluble Klotho can be found in the blood, cerebrospinal fluid, and the urine of mammals. Klotho deficiency results in early appearance of multiple age-related disorders and premature death, whereas overexpression of Klotho exerts the opposite effect. Klotho may influence cellular transport processes across the cell membrane by inhibiting calcitriol (1,25(OH) (2)D(3)), formation or by directly affecting transporter proteins, including ion channels, carriers and pumps. Accordingly, Klotho protein is a powerful regulator of transport mechanisms across the cell membrane. Klotho regulates diverse calcium and potassium ion channels, as well as several carriers including the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na(+)-coupled phosphate cotransporters, NaPi-IIa and NaPi-IIb, and a Na(+)/K(+)-ATPase. All those cellular transport regulations contribute in the aging suppressor role of Klotho. Future studies will help to determine if the Klotho protein regulates cell-surface expression of other transport proteins and is affecting underlying mechanisms.

  5. Structure-function analysis of the retinoblastoma tumor suppressor protein – is the whole a sum of its parts?

    Directory of Open Access Journals (Sweden)

    Dick Frederick A

    2007-09-01

    Full Text Available Abstract Biochemical analysis of the retinoblastoma protein's function has received considerable attention since it was cloned just over 20 years ago. During this time pRB has emerged as a key regulator of the cell division cycle and its ability to block proliferation is disrupted in the vast majority of human cancers. Much has been learned about the regulation of E2F transcription factors by pRB in the cell cycle. However, many questions remain unresolved and researchers continue to explore this multifunctional protein. In particular, understanding how its biochemical functions contribute to its role as a tumor suppressor remains to be determined. Since pRB has been shown to function as an adaptor molecule that links different proteins together, or to particular promoters, analyzing pRB by disrupting individual protein interactions holds tremendous promise in unraveling the intricacies of its function. Recently, crystal structures have reported how pRB interacts with some of its molecular partners. This information has created the possibility of rationally separating pRB functions by studying mutants that disrupt individual binding sites. This review will focus on literature that investigates pRB by isolating functions based on binding sites within the pocket domain. This article will also discuss the prospects for using this approach to further explore the unknown functions of pRB.

  6. Nuclear pore component Nup98 is a potential tumor suppressor and regulates posttranscriptional expression of select p53 target genes.

    Science.gov (United States)

    Singer, Stephan; Zhao, Ruiying; Barsotti, Anthony M; Ouwehand, Anette; Fazollahi, Mina; Coutavas, Elias; Breuhahn, Kai; Neumann, Olaf; Longerich, Thomas; Pusterla, Tobias; Powers, Maureen A; Giles, Keith M; Leedman, Peter J; Hess, Jochen; Grunwald, David; Bussemaker, Harmen J; Singer, Robert H; Schirmacher, Peter; Prives, Carol

    2012-12-14

    The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3σ) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. The tumor suppressor Rb and its related Rbl2 genes are regulated by Utx histone demethylase

    Energy Technology Data Exchange (ETDEWEB)

    Terashima, Minoru; Ishimura, Akihiko; Yoshida, Masakazu [Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Ishikawa (Japan); Suzuki, Yutaka; Sugano, Sumio [Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Chiba (Japan); Suzuki, Takeshi, E-mail: suzuki-t@staff.kanazawa-u.ac.jp [Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Ishikawa (Japan)

    2010-08-20

    Research highlights: {yields} Utx increases expression of Rb and Rbl2 genes through its demethylase activity. {yields} Utx changes histone H3 methylation on the Rb and Rbl2 promoters. {yields} Utx induces decreased cell proliferation of mammalian primary cells. -- Abstract: Utx is a candidate tumor suppressor gene that encodes histone H3 lysine 27 (H3K27) demethylase. In this study, we found that ectopic expression of Utx enhanced the expression of retinoblastoma tumor suppressor gene Rb and its related gene Rbl2. This activation was dependent on the demethylase activity of Utx, and was suggested to contribute to the decreased cell proliferation induced by Utx. A chromatin immunoprecipitation assay showed that over-expressed Utx was associated with the promoter regions of Rb and Rbl2 resulting in the removal of repressive H3K27 tri-methylation and the increase in active H3K4 tri-methylation. Furthermore, siRNA-mediated knockdown of Utx revealed the recruitment of endogenous Utx protein on the promoters of Rb and Rbl2 genes. These results indicate that Rb and Rbl2 are downstream target genes of Utx and may play important roles in Utx-mediated cell growth control.

  8. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  9. The von Hippel-Lindau tumor suppressor regulates programmed cell death 5-mediated degradation of Mdm2

    NARCIS (Netherlands)

    Essers, P B; Klasson, T D; Pereboom, T C; Mans, D A; Nicastro, M; Boldt, K; Giles, R H; MacInnes, A W

    2015-01-01

    Functional loss of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), which is part of an E3-ubiquitin ligase complex, initiates most inherited and sporadic clear-cell renal cell carcinomas (ccRCC). Genetic inactivation of the TP53 gene in ccRCC is rare, suggesting that an alternate

  10. Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity.

    Science.gov (United States)

    Mathur, Kalika; Anand, Abhishek; Dubey, Sunil Kumar; Sanan-Mishra, Neeti; Bhatnagar, Raj K; Sunil, Sujatha

    2016-11-30

    RNAi pathway is an antiviral defence mechanism employed by insects that result in degradation of viral RNA thereby curbing infection. Several viruses including flaviviruses encode viral suppressors of RNAi (VSRs) to counteract the antiviral RNAi pathway. Till date, no VSR has been reported in alphaviruses. The present study was undertaken to evaluate chikungunya virus (CHIKV) proteins for RNAi suppressor activity. We systematically analyzed all nine CHIKV proteins for RNAi suppressor activity using Sf21 RNAi sensor cell line based assay. Two non-structural proteins, namely, nsP2 and nsP3 were found to exhibit RNAi suppressor activity. We further validated the findings in natural hosts, namely in Aedes and in mammalian cell lines and further through EMSA and Agrobacterium infiltration in GFP silenced transgenic tobacco plants. Domains responsible for maximum RNAi suppressor activity were also identified within these proteins. RNA binding motifs in these domains were identified and their participation in RNAi suppression evaluated using site directed mutagenesis. Sequence alignment of these motifs across all species of known alphaviruses revealed conservation of these motifs emphasizing on a similar role of action in other species of alphaviruses as well. Further validation of RNAi suppressor activity of these proteins awaits establishment of specific virus infection models.

  11. Down-regulation of SFRP1 as a putative tumor suppressor gene can contribute to human hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Huang, Jian; Zhang, Yun-Li; Teng, Xiao-Mei; Lin, Yun; Zheng, Da-Li; Yang, Peng-Yuan; Han, Ze-Guang

    2007-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. SFRP1 (the secreted frizzled-related protein 1), a putative tumor suppressor gene mapped onto chromosome 8p12-p11.1, the frequent loss of heterozygosity (LOH) region in human HCC, encodes a Wingless-type (Wnt) signaling antagonist and is frequently inactivated by promoter methylation in many human cancers. However, whether the down-regulation of SFRP1 can contribute to hepatocarcinogenesis still remains unclear. We investigated the expression of SFRP1 through real time RT-PCR and immunohistochemistry staining. The cell growth and colony formation were observed as the overexpression and knockdown of SFRP1. The DNA methylation status within SFRP1 promoter was analyzed through methylation-specific PCR or bisulphate-treated DNA sequencing assays. Loss of heterozygosity was here detected with microsatellite markers. SFRP1 was significantly down-regulated in 76.1% (35/46) HCC specimens at mRNA level and in 30% (30/100) HCCs indicated by immunohistochemistry staining, as compared to adjacent non-cancerous livers. The overexpression of SFRP1 can significantly inhibit the cell growth and colony formation of YY-8103, SMMC7721, and Hep3B cells. The RNA interference against the constitutional SFRP1 in the offspring SMMC7721 cells, which were stably transfected by ectopic SFRP1, can markedly promote cell growth of these cells. LOH of both microsatellite markers D8S532 and D8SAC016868 flanking the gene locus was found in 13% (6 of 46 HCCs) and 6.5% (3 of 46 HCCs) of the informative cases, respectively, where 5 of 8 HCC specimens with LOH showed the down-regulation of SFRP1. DNA hypermethylation within SFRP1 promoter was identified in two of three HCC specimens without SFRP1 expression. Moreover, the DNA methylation of SFRP1 promoter was significantly reduced, along with the re-expression of the gene, in those HCC cell lines, Bel7404, QGY7701, and MHCC-H, as treated by DAC. Our data suggested that the

  12. The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

    Directory of Open Access Journals (Sweden)

    Corinne Besnard-Guérin

    Full Text Available Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs. Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2. Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

  13. The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

    Science.gov (United States)

    Fusaro, Adriana F; Barton, Deborah A; Nakasugi, Kenlee; Jackson, Craig; Kalischuk, Melanie L; Kawchuk, Lawrence M; Vaslin, Maite F S; Correa, Regis L; Waterhouse, Peter M

    2017-10-10

    The plant viral family Luteoviridae is divided into three genera: Luteovirus , Polerovirus and Enamovirus . Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

  14. Are suppressors of cytokine signaling proteins recently identified in atherosclerosis possible therapeutic targets?

    Science.gov (United States)

    Tang, Jingjing; Raines, Elaine W

    2005-10-01

    Atherosclerosis is a slowly progressing chronic inflammatory disease characterized by focal arterial lesions that can ultimately occlude the entire blood vessel and lead to sudden death. Lesions associated with cardiovascular events are those enriched in macrophages and other inflammatory cells. Activation of inflammatory cells within lesions induces the release of cytokines which promotes more inflammation and associated tissue damage if cytokine signaling pathways remain unregulated. Thus, pathways capable of suppressing proinflammatory cytokine signaling hold the potential to limit life-threatening cardiovascular events caused by atherogenesis. This review focuses on suppressors of cytokine signaling proteins recently identified in the atherosclerosis-prone ApoE(-/-) mouse and provides perspectives of their potential for intervention in atherosclerotic lesion progression.

  15. MTUS1 tumor suppressor and its miRNA regulators in fibroadenoma and breast cancer.

    Science.gov (United States)

    Kara, Murat; Kaplan, Mehmet; Bozgeyik, Ibrahim; Ozcan, Onder; Celik, Ozgur Ilhan; Bozgeyik, Esra; Yumrutas, Onder

    2016-08-10

    Breast cancer is major public health problem predominantly effects female population. Current therapeutic approaches to deal with breast cancer are still lack of effectiveness. Thus, identifying/developing novel strategies to fight against breast cancer is very important. The frequent deletions at 8p21.3-22 chromosomal location nearby D8S254 marker enabled the discovery of a novel tumor suppressor gene, MTUS1. Subsequently, MTUS1 was demonstrated to be less expressed in a variety cancer types including breast cancer. Also, it is obvious that gene expression is widely regulated by miRNAs. Here, we aimed to report differential expression of MTUS1 and its regulatory miRNAs in breast cancer and fibroadenoma tissues. Dynamic analysis of MTUS1 expression levels and its miRNAs regulators were attained by Fluidigm 96×96 Dynamic Array Expression chips and reactions were performed in Fluidigm BioMark™ HD System qPCR. Consequently, MTUS1 mRNA levels were significantly diminished in breast cancer tissues and elevated in fibroadenoma tissues. Also, among MTUS1 targeting miRNAs, miR-183-5p was identified to be overexpressed in breast cancer and down-regulated in fibroadenoma tissues. Also, expression levels of MTUS1 and miR-183-5p were well correlated with clinical parameters. In particular, MTUS1 expression was found to be diminished and miR-183-5p expression was elevated with the advancing stage. In conclusion, as a potential therapeutic target, miR-183-5p can be a chief regulator of MTUS1 and MTUS1-miR-183-5p axis may have significant influence in the pathology of breast cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

    Science.gov (United States)

    Baxley, Ryan M.; Soshnev, Alexey A.; Koryakov, Dmitry E.; Zhimulev, Igor F.; Geyer, Pamela K.

    2011-01-01

    The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190 kD (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions. PMID:21651900

  17. Tumor suppressor p53 negatively regulates glycolysis stimulated by hypoxia through its target RRAD

    Science.gov (United States)

    Wu, Rui; Liang, Yingjian; Lin, Meihua; Liu, Jia; Chan, Chang S.; Hu, Wenwei; Feng, Zhaohui

    2014-01-01

    Cancer cells display enhanced glycolysis to meet their energetic and biosynthetic demands even under normal oxygen concentrations. Recent studies have revealed that tumor suppressor p53 represses glycolysis under normoxia as a novel mechanism for tumor suppression. As the common microenvironmental stress for tumors, hypoxia drives the metabolic switch from the oxidative phosphorylation to glycolysis, which is crucial for survival and proliferation of cancer cells under hypoxia. The p53's role and mechanism in regulating glycolysis under hypoxia is poorly understood. Here, we found that p53 represses hypoxia-stimulated glycolysis in cancer cells through RRAD, a newly-identified p53 target. RRAD expression is frequently decreased in lung cancer. Ectopic expression of RRAD greatly reduces glycolysis whereas knockdown of RRAD promotes glycolysis in lung cancer cells. Furthermore, RRAD represses glycolysis mainly through inhibition of GLUT1 translocation to the plasma membrane. Under hypoxic conditions, p53 induces RRAD, which in turn inhibits the translocation of GLUT1 and represses glycolysis in lung cancer cells. Blocking RRAD by siRNA greatly abolishes p53's function in repressing glycolysis under hypoxia. Taken together, our results revealed an important role and mechanism of p53 in antagonizing the stimulating effect of hypoxia on glycolysis, which contributes to p53's function in tumor suppression. PMID:25114038

  18. Immunocytochemical mapping of the phosphatase and tensin homolog (PTEN/MMAC1) tumor suppressor protein in human gliomas.

    OpenAIRE

    Fults, D.; Pedone, C.

    2000-01-01

    PTEN/MMAC1 (phosphatase and tensin homolog/mutated in multiple advanced cancers 1) is a tumor suppressor gene, the inactivation of which is an important step in the progression of gliomas to end-stage glioblastoma multiforme. We examined the distribution of PTEN protein in 49 primary human gliomas by immunocytochemistry using polyclonal antibodies that we raised against PTEN-glutathione S-transferase fusion proteins expressed in Escherichia coli. The study group consisted of 6 low-grade astro...

  19. The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity.

    Directory of Open Access Journals (Sweden)

    Ruilin Wang

    Full Text Available Cucumber mosaic virus (CMV is a model virus for plant-virus protein interaction and mechanism research because of its wide distribution, high-level of replication and simple genome structure. The 2b protein is a multifunctional protein encoded by CMV that suppresses RNA silencing-based antiviral defense and contributes to CMV virulence in host plants. In this report, 12 host proteins were identified as CMV LS2b binding partners using the yeast two-hybrid screen system from the Arabidopsis thaliana cDNA library. Among the host proteins, 30S ribosomal subunit protein S11 (RPS11 was selected for further studies. The interaction between LS2b and full-length RPS11 was confirmed using the yeast two-hybrid system. Bimolecular fluorescence complementation (BIFC assays observed by confocal laser microscopy and Glutathione S-transferase (GST pull-down assays were used to verify the interaction between endogenous NbRPS11 and viral CMVLS2b both in vivo and in vitro. TRV-based gene silencing vector was used to knockdown NbRPS11 transcription, and immunoblot analysis revealed a decline in infectious viral RNA replication and a decrease in CMV infection in RPS11 down-regulated Nicotiana benthamiana plants. Thus, the knockdown of RPS11 likely inhibited CMV replication and accumulation. The gene silencing suppressor activity of CMV2b protein was reduced by the RPS11 knockdown. This study demonstrated that the function of viral LS2b protein was remarkably affected by the interaction with host RPS11 protein.

  20. The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing

    Directory of Open Access Journals (Sweden)

    Adriana F. Fusaro

    2017-10-01

    Full Text Available The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant’s vascular system. The first open reading frame (ORF of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs against the plant’s viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV, however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant’s silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant’s anti-viral defense.

  1. The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

    Science.gov (United States)

    Hsu, Shih-Jui; Plata, Maria P; Ernest, Ben; Asgarifar, Saghi; Labrador, Mariano

    2015-07-01

    Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

    Science.gov (United States)

    Chen, Muhan; Nowak, Dawid G; Narula, Navneet; Robinson, Brian; Watrud, Kaitlin; Ambrico, Alexandra; Herzka, Tali M; Zeeman, Martha E; Minderer, Matthias; Zheng, Wu; Ebbesen, Saya H; Plafker, Kendra S; Stahlhut, Carlos; Wang, Victoria M Y; Wills, Lorna; Nasar, Abu; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Wilkinson, John E; Powers, Scott; Sordella, Raffaella; Altorki, Nasser K; Mittal, Vivek; Stiles, Brendon M; Plafker, Scott M; Trotman, Lloyd C

    2017-03-06

    Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele. © 2017 Chen et al.

  3. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

    DEFF Research Database (Denmark)

    Nguyen, Thanh Hung; Kugler, Jan-Michael; Cohen, Stephen Michael

    2017-01-01

    /TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family...... deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor...

  4. Post-transcriptional regulation of the tumor suppressor p53 by a novel miR-27a, with implications during hypoxia and tumorigenesis.

    Science.gov (United States)

    Maqbool, Raihana; Lone, Saife Niaz; Ul Hussain, Mahboob

    2016-10-15

    The tumor suppressor protein p53 is intricately regulated by various signaling molecules, including non-coding small RNAs, called microRNAs (miRNAs). The in silico analysis and the inverse expression status in various cell lines raised the possibility of miR-27a being a new regulator of p53. Using luciferase reporter assay and various mutational and functional analysis, we identified two putative binding sites of miR-27a on the 3'-UTR of p53. The overexpression of miR-27a in the human colorectal cancer cell line HCT-116 +/+ resulted in the decreased expression of the endogenous p53 protein levels. During hypoxia of the HCT-116 +/+ cells, p53 showed increased accumulation after 3 h, and the levels were significantly up-regulated until 24 h of hypoxia. The p53 expression dynamics during hypoxia of the HCT-116 +/+ cells were found to be inversely regulated by miR-27a expression. Moreover, using a cell viability assay, we established that after 3 h of hypoxia, the accumulation of p53 results in a decreased number of the viable HCT-116 +/+ cells and the overexpression of miR-27a resulted in an increased number of viable HCT-116 +/+ cells with a concomitant decrease in p53 expression. Additionally, our data indicated that miR-27a and p53 depict inverse expression dynamics in 50% of the human colorectal cancer samples studied, when compared with that in the adjacent normal samples. Our data established that miR-27a and the tumor suppressor protein p53 are part of the same signaling network that has important implications during hypoxia and tumorigenesis. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  5. Redox regulation of the tumor suppressor PTEN by the thioredoxin system and cumene hydroperoxide.

    Science.gov (United States)

    Han, Seong-Jeong; Zhang, Ying; Kim, Inyoung; Chay, Kee-Oh; Yoon, Hyun Joong; Jang, Dong Il; Yang, Sung Yeul; Park, Jiyoung; Woo, Hyun Ae; Park, Iha; Lee, Seung-Rock

    2017-11-01

    Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1)

    Energy Technology Data Exchange (ETDEWEB)

    Yates, Luke A., E-mail: luke@strubi.ox.ac.uk; Durrant, Benjamin P.; Barber, Michael; Harlos, Karl [University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Fleurdépine, Sophie; Norbury, Chris J. [University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Gilbert, Robert J. C., E-mail: luke@strubi.ox.ac.uk [University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2015-02-21

    The use of truncation and RNA-binding mutations of caffeine induced death suppressor protein 1 (Cid1) as a means to enhance crystallogenesis leading to an improvement of X-ray diffraction resolution by 1.5 Å is reported. The post-transcriptional addition of uridines to the 3′-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3′-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate microRNAs, altering their maturation. Cid1 has been studied as a model TUT that has provided insights into the larger and more complex metazoan enzyme system. In this paper, two strategies are described that led to improvements both in the crystallogenesis of Cid1 and in the resolution of diffraction by ∼1.5 Å. These advances have allowed high-resolution crystallo@@graphic studies of this TUT system to be initiated.

  7. The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor

    Science.gov (United States)

    Bates, Gaynor J; Nicol, Samantha M; Wilson, Brian J; Jacobs, Anne-Marie F; Bourdon, Jean-Christophe; Wardrop, Julie; Gregory, David J; Lane, David P; Perkins, Neil D; Fuller-Pace, Frances V

    2005-01-01

    The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-κB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity. PMID:15660129

  8. Candidate tumour suppressor CCDC19 regulates miR-184 direct targeting of C-Myc thereby suppressing cell growth in non-small cell lung cancers.

    Science.gov (United States)

    Liu, Zhen; Mai, Chunping; Yang, Huiling; Zhen, Yan; Yu, Xiaoli; Hua, Shengni; Wu, Qiangyun; Jiang, Qinping; Zhang, Yajie; Song, Xin; Fang, Weiyi

    2014-08-01

    We previously reported and revised the nasopharyngeal epithelium specific protein CCDC19 and identified it as a potential tumour suppressor in nasopharyngeal carcinoma. The purpose of this study was to investigate the involvement of CCDC19 in the pathogenesis of human non-small cell lung cancers (NSCLC). Down-regulated CCDC19 expression was observed in NSCLC tissues and cells compared to normal tissues. However, reduced protein expression did not correlate with the status of NSCLC progression. Instead, we observed that patients with lower CCDC19 expression had a shorter overall survival than did patients with higher CCDC19 expression. Lentiviral-mediated CCDC19 overexpression significantly suppressed cell proliferation and cell cycle transition from G1 to S and G2 phases in NSCLC cells. Knocking down CCDC19 expression significantly restored the ability of cell growth in CCDC19 overexpressing NSCLC cells. Mechanistically CCDC19 functions as a potential tumour suppressor by stimulating miR-184 suppression of C-Myc thus blocking cell growth mediated by the PI3K/AKT/C-Jun pathway. Our studies are the first to demonstrate that reduced expression of CCDC19 is an unfavourable factor in NSCLC. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  9. 99: A Novel Myc-Interacting Protein with Features of a Breast Tumor Suppressor Gene Product

    National Research Council Canada - National Science Library

    Prendergast, George

    1997-01-01

    Bin1 is a novel tumor suppressor-like molecule we identified through its ability to interact with and inhibit the oncogenic activity of the Myc oncoprotein, which is widely deregulated in breast cancer...

  10. Evidence that selenium binding protein 1 is a tumor suppressor in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Emmanuel Ansong

    Full Text Available Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56 is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.

  11. Evidence that selenium binding protein 1 is a tumor suppressor in prostate cancer.

    Science.gov (United States)

    Ansong, Emmanuel; Ying, Qi; Ekoue, Dede N; Deaton, Ryan; Hall, Andrew R; Kajdacsy-Balla, Andre; Yang, Wancai; Gann, Peter H; Diamond, Alan M

    2015-01-01

    Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.

  12. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

    Energy Technology Data Exchange (ETDEWEB)

    Fusaro, Adriana F. [University of Sydney, NSW 2006 (Australia); CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia); Correa, Regis L. [CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia); Depto. de Virologia, IMPPG, UFRJ, 21941-902 (Brazil); Nakasugi, Kenlee; Jackson, Craig [University of Sydney, NSW 2006 (Australia); Kawchuk, Lawrence [Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J4B1 (Canada); Vaslin, Maite F.S. [Depto. de Virologia, IMPPG, UFRJ, 21941-902 (Brazil); Waterhouse, Peter M., E-mail: peter.waterhouse@sydney.edu.au [University of Sydney, NSW 2006 (Australia); CSIRO Plant Industry, Canberra, P.O. Box 1600, ACT 2601 (Australia)

    2012-05-10

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

  13. Relevance of miR-21 in regulation of tumor suppressor gene PTEN in human cervical cancer cells

    International Nuclear Information System (INIS)

    Peralta-Zaragoza, Oscar; Deas, Jessica; Meneses-Acosta, Angélica; De la O-Gómez, Faustino; Fernández-Tilapa, Gloria; Gómez-Cerón, Claudia; Benítez-Boijseauneau, Odelia; Burguete-García, Ana; Torres-Poveda, Kirvis; Bermúdez-Morales, Victor Hugo; Madrid-Marina, Vicente; Rodríguez-Dorantes, Mauricio; Hidalgo-Miranda, Alfredo; Pérez-Plasencia, Carlos

    2016-01-01

    Expression of the microRNA miR-21 has been found to be altered in almost all types of cancers and it has been classified as an oncogenic microRNA or oncomir. Due to the critical functions of its target proteins in various signaling pathways, miR-21 is an attractive target for genetic and pharmacological modulation in various cancers. Cervical cancer is the second most common cause of death from cancer in women worldwide and persistent HPV infection is the main etiologic agent. This malignancy merits special attention for the development of new treatment strategies. In the present study we analyze the role of miR-21 in cervical cancer cells. To identify the downstream cellular target genes of upstream miR-21, we silenced endogenous miR-21 expression in a cervical intraepithelial neoplasia-derived cell lines using siRNAs. The effect of miR-21 on gene expression was assessed in cervical cancer cells transfected with the siRNA expression plasmid pSIMIR21. We identified the tumor suppressor gene PTEN as a target of miR-21 and determined the mechanism of its regulation throughout reporter construct plasmids. Using this model, we analyzed the expression of miR-21 and PTEN as well as functional effects such as autophagy and apoptosis induction. In SiHa cells, there was an inverse correlation between miR-21 expression and PTEN mRNA level as well as PTEN protein expression in cervical cancer cells. Transfection with the pSIMIR21 plasmid increased luciferase reporter activity in construct plasmids containing the PTEN-3′-UTR microRNA response elements MRE21-1 and MRE21-2. The role of miR-21 in cell proliferation was also analyzed in SiHa and HeLa cells transfected with the pSIMIR21 plasmid, and tumor cells exhibited markedly reduced cell proliferation along with autophagy and apoptosis induction. We conclude that miR-21 post-transcriptionally down-regulates the expression of PTEN to promote cell proliferation and cervical cancer cell survival. Therefore, it may be a

  14. Primary microcephaly gene MCPH1 shows signatures of tumor suppressors and is regulated by miR-27a in oral squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Thejaswini Venkatesh

    Full Text Available Mutations in the MCPH1 (microcephalin 1 gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC samples, and observed that 14/71 (19.72% informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22% and 19/25 (76% OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10% tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

  15. SUMOylation of the ING1b tumor suppressor regulates gene transcription

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Guérillon, Claire; Kim, Tae-Sun

    2014-01-01

    and senescence, but how the proteins themselves are regulated is not yet clear. Here, we find a small ubiquitin-like modification (SUMOylation) of the ING1b protein and identify lysine 193 (K193) as the preferred ING1b SUMO acceptor site. We also show that PIAS4 is the E3 SUMO ligase responsible for ING1b...... SUMOylation on K193. Sequence alignment reveals that the SUMO consensus site on ING1b contains a phosphorylation-dependent SUMOylation motif (PDSM) and our data indicate that the SUMOylation on K193 is enhanced by the S199D phosphomimic mutant. Using an ING1b protein mutated at the major SUMOylation site (ING...

  16. Significance of expression of suppressor of cytokine signaling proteins: Suppressor of cytokine signaling-1, suppressor of cytokine signaling-2, and suppressor of cytokine signaling-3 in papillary thyroid cancer

    Directory of Open Access Journals (Sweden)

    Toral Pundrik Kobawala

    2017-01-01

    Conclusion: Expression of the studied SOCS proteins may be a consequence of activation of Janus kinase-signal transducers and activators of transcription and other pathways supporting growth and survival of cancer cells that are sustained by several cytokines. Thus, SOCS-1, SOCS-2, and SOCS-3 proteins may directly or indirectly, have important roles in development and pathogenesis of papillary thyroid cancer.

  17. Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene

    OpenAIRE

    Gil, Elad B.; Malone Link, Elizabeth; Liu, Leo X.; Johnson, Carl D.; Lees, Jacqueline A.

    1999-01-01

    The human PTEN tumor suppressor gene is mutated in a wide variety of sporadic tumors. To determine the function of PTEN in vivo we have studied a PTEN homolog in Caenorhabditis elegans. We have generated a strong loss-of-function allele of the PTEN homolog and shown that the deficient strain is unable to enter dauer diapause. An insulin-like phosphatidylinositol 3-OH kinase (PI3′K) signaling pathway regulates dauer-stage entry. Mutations in either the daf-2 insulin receptor-like (IRL) gene or...

  18. VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex.

    Science.gov (United States)

    Zhang, Wenjing; Gao, Yijun; Li, Peixue; Shi, Zhubing; Guo, Tong; Li, Fei; Han, Xiangkun; Feng, Yan; Zheng, Chao; Wang, Zuoyun; Li, Fuming; Chen, Haiquan; Zhou, Zhaocai; Zhang, Lei; Ji, Hongbin

    2014-03-01

    Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

  19. Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

    Science.gov (United States)

    Naim, Fatima; Shrestha, Pushkar; Singh, Surinder P; Waterhouse, Peter M; Wood, Craig C

    2016-06-01

    Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  20. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    triguingly, the substrate or the product of the inhibited enzyme can be structurally different from the inhibitor. ... ulation of proteins in this fashion as 'allosteric' in the year 1961. [9]. The word allostery originated from the ..... flux occurs via the conformational selec- tion pathway at low concentrations of the ligand, while the trend.

  1. Suppressor of cytokine signaling (SOCS genes are silenced by DNA hypermethylation and histone deacetylation and regulate response to radiotherapy in cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Moon-Hong Kim

    Full Text Available Suppressor of cytokine signaling (SOCS family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.

  2. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    Energy Technology Data Exchange (ETDEWEB)

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Katiyar, Santosh K., E-mail: skatiyar@uab.edu [Birmingham Veterans Affairs Medical Center, Birmingham, AL 35294 (United States); Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2012-08-15

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16{sup INK4a} and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. -- Highlights: ►Epigenetic modulations have been shown to have a role in cancer risk. ►Proanthocyanidins decrease the levels of DNA methylation and histone deacetylation. ►Proanthocyanidins inhibit histone deacetylase activity in skin cancer cells. ►Proanthocyanidins reactivate tumor suppressor genes in skin

  3. Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

    International Nuclear Information System (INIS)

    Vaid, Mudit; Prasad, Ram; Singh, Tripti; Jones, Virginia; Katiyar, Santosh K.

    2012-01-01

    Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16 INK4a and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans. -- Highlights: ►Epigenetic modulations have been shown to have a role in cancer risk. ►Proanthocyanidins decrease the levels of DNA methylation and histone deacetylation. ►Proanthocyanidins inhibit histone deacetylase activity in skin cancer cells. ►Proanthocyanidins reactivate tumor suppressor genes in skin

  4. Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1.

    Science.gov (United States)

    Benzeno, Sharon; Narla, Goutham; Allina, Jorge; Cheng, George Z; Reeves, Helen L; Banck, Michaela S; Odin, Joseph A; Diehl, J Alan; Germain, Doris; Friedman, Scott L

    2004-06-01

    Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.

  5. SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT.

    Directory of Open Access Journals (Sweden)

    Praveen Bhoopathi

    Full Text Available Secreted protein acidic and rich in cysteine (SPARC is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.

  6. The tumor suppressors pRB and p53 as regulators of adipocyte differentiation and function

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Feddersen, Søren; Madsen, Lise

    2009-01-01

    BACKGROUND: The retinoblastoma protein (pRB) and p53 are crucial members of regulatory networks controlling the cell cycle and apoptosis, and a hallmark of virtually all cancers is dysregulation of expression or function of pRB or p53. Although they are best known for their role in cancer...... development, it is now evident that both are implicated in metabolism and cellular development. OBJECTIVE/METHODS: To review the role of pRB and p53 in adipocyte differentiation and function emphasizing that pRB and p53, via their effects on adipocyte development and function, play a role in the regulation...... of energy metabolism and homeostasis. RESULTS/CONCLUSIONS: pRB is required for adipose conversion and also involved in determining its mitochondrial capacity. p53 inhibits adipogenesis and results suggest that it is involved in maintaining function of adipose tissue....

  7. Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

    Science.gov (United States)

    Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

    2016-11-29

    Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

  8. Restoring Sensitivity to Apoptosis in Prostate Cancer Cells by Reconstitution of the Tumor Suppressor PTEN

    National Research Council Canada - National Science Library

    Whang, Young

    2003-01-01

    ... suppressor PTEN in regulating sensitivity to apoptosis in prostate cancer. We have previously shown that loss of HEN function leads to excessive antiapoptotic signaling through constitutive activation of the Akt protein kinase...

  9. Regulation of Ubiquitination-Mediated Protein Degradation by Survival Kinases in Cancer

    International Nuclear Information System (INIS)

    Yamaguchi, Hirohito; Hsu, Jennifer L.; Hung, Mien-Chie

    2012-01-01

    The ubiquitin–proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, AKT, extracellular signal-regulated kinase, and IκB kinase, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, myeloid cell leukemia-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic.

  10. Stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells.

    Science.gov (United States)

    Padmanabhan, Achuth; Gosc, Eliza B; Bieberich, Charles J

    2013-05-01

    Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells. Copyright © 2012 Wiley Periodicals, Inc.

  11. Blood Serum Alpha Fetoprotein Enhancer Binding Protein, a Tumor Suppressor, Decreases in Chronic HBV Hepatitis Patients as Hepatocellular Cancer Appears

    Directory of Open Access Journals (Sweden)

    James N. Riggins

    2010-01-01

    Full Text Available Chronic hepatitis increases the risk of hepatocellular carcinoma (HCC. To test whether circulating proteins reflect hepatic carcinogenesis, sera from patients and controls were albumin depleted, enriched for glycoproteins, digested with trypsin, and subjected to reverse phase chromatography and tandem mass spectrometry. Alpha-fetoprotein enhancer binding protein (AFPebp, a tumor suppressor, was repeatedly identified in sera from chronic HBV hepatitis patients. We independently identified and quantified AFPebp with a deuterated, phenylisocyanate-labeled synthetic peptide standard. Elevated AFPebp levels in sera from chronic HBV hepatitis patients decreased as cancer developed. These data suggest that rising AFPebp levels in chronic HBV hepatitis may be protective, while falling levels may contribute to HCC development.

  12. Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade

    Directory of Open Access Journals (Sweden)

    Alessi Dario R

    2003-09-01

    Full Text Available Abstract Background The AMP-activated protein kinase (AMPK cascade is a sensor of cellular energy charge that acts as a 'metabolic master switch' and inhibits cell proliferation. Activation requires phosphorylation of Thr172 of AMPK within the activation loop by upstream kinases (AMPKKs that have not been identified. Recently, we identified three related protein kinases acting upstream of the yeast homolog of AMPK. Although they do not have obvious mammalian homologs, they are related to LKB1, a tumor suppressor that is mutated in the human Peutz-Jeghers cancer syndrome. We recently showed that LKB1 exists as a complex with two accessory subunits, STRADα/β and MO25α/β. Results We report the following observations. First, two AMPKK activities purified from rat liver contain LKB1, STRADα and MO25α, and can be immunoprecipitated using anti-LKB1 antibodies. Second, both endogenous and recombinant complexes of LKB1, STRADα/β and MO25α/β activate AMPK via phosphorylation of Thr172. Third, catalytically active LKB1, STRADα or STRADβ and MO25α or MO25β are required for full activity. Fourth, the AMPK-activating drugs AICA riboside and phenformin do not activate AMPK in HeLa cells (which lack LKB1, but activation can be restored by stably expressing wild-type, but not catalytically inactive, LKB1. Fifth, AICA riboside and phenformin fail to activate AMPK in immortalized fibroblasts from LKB1-knockout mouse embryos. Conclusions These results provide the first description of a physiological substrate for the LKB1 tumor suppressor and suggest that it functions as an upstream regulator of AMPK. Our findings indicate that the tumors in Peutz-Jeghers syndrome could result from deficient activation of AMPK as a consequence of LKB1 inactivation.

  13. Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation.

    Science.gov (United States)

    Iijima, Issei; Hohsaka, Takahiro

    2009-01-01

    Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.

  14. Cellular regulation by protein phosphorylation.

    Science.gov (United States)

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. Copyright © 2012. Published by Elsevier Inc.

  15. Host natural suppressor activity regulates hemopoietic engraftment kinetics in antibody-conditioned recipient mice

    International Nuclear Information System (INIS)

    Sadelain, M.W.; Green, D.R.; Wegmann, T.G.

    1990-01-01

    Resistance to semi-allogeneic or syngeneic hemopoietic stem cell engraftment can be reduced by treating the unirradiated host with anti-class I MHC antibody. In our previous studies we showed a direct correlation between such resistance and the level of natural suppressor (NS) activity in the host. Thus newborn mice that have high NS activity are very resistant to marrow engraftment, as are adults pretreated with CFA that increases NS activity in the bone marrow. We have now devised a method that allows us to follow hemopoietic engraftment kinetics within the marrow cavity itself by assaying individual CFU-granulocyte/macrophage progenitor cells for their host or donor origin over the immediate post-transplant period. By using this method, we find a close correlation between the rate of marrow engraftment and reduction in host NS activity. Marrow engraftment does not correlate with the reduction of either total host bone marrow cellular content or CFU-granulocyte/macrophage progenitor cell levels. NS activity is mediated by Thy-1-, partially radiosensitive, nylon wool nonadherent cells without NK activity. Adoptively transferred Thy-1-, irradiated spleen cells containing NS activity induced by pretreatment with CFA delayed engraftment kinetics in the marrow cavity. Thus hemopoietic engraftment in the marrow cavity appears to be controlled by an inhibitory regulatory activity that is reflected in the in vitro NS assay. These studies suggest new regulatory targets for selective host conditioning to eliminate resistance to marrow transplantation

  16. The tumor suppressor PTEN and the PDK1 kinase regulate formation of the columnar neural epithelium

    Science.gov (United States)

    Grego-Bessa, Joaquim; Bloomekatz, Joshua; Castel, Pau; Omelchenko, Tatiana; Baselga, José; Anderson, Kathryn V

    2016-01-01

    Epithelial morphogenesis and stability are essential for normal development and organ homeostasis. The mouse neural plate is a cuboidal epithelium that remodels into a columnar pseudostratified epithelium over the course of 24 hr. Here we show that the transition to a columnar epithelium fails in mutant embryos that lack the tumor suppressor PTEN, although proliferation, patterning and apical-basal polarity markers are normal in the mutants. The Pten phenotype is mimicked by constitutive activation of PI3 kinase and is rescued by the removal of PDK1 (PDPK1), but does not depend on the downstream kinases AKT and mTORC1. High resolution imaging shows that PTEN is required for stabilization of planar cell packing in the neural plate and for the formation of stable apical-basal microtubule arrays. The data suggest that appropriate levels of membrane-associated PDPK1 are required for stabilization of apical junctions, which promotes cell elongation, during epithelial morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.12034.001 PMID:26809587

  17. Regulation of ubiquitination-mediated protein degradation by survival kinases in cancer

    Directory of Open Access Journals (Sweden)

    Hirohito eYamaguchi

    2012-02-01

    Full Text Available The ubiquitin-proteasome system is essential for multiple physiological processes via selective degradation of target proteins and has been shown to plays a critical role in human cancer. Activation of oncogenic factors and inhibition of tumor suppressors have been shown to be essential for cancer development, and protein ubiquitination has been linked to the regulation of oncogenic factors and tumor suppressors. Three kinases, Akt, ERK, and IKK, we refer to as oncokinases, are activated in multiple human cancers. We and others have identified several key downstream targets that are commonly regulated by these oncokinases, some of which are regulated directly or indirectly via ubiquitin-mediated proteasome degradation, including FOXO3, β-catenin, Mcl-1, and Snail. In this review, we summarize these findings from our and other groups and discuss potential future studies and applications in the clinic.

  18. Suppressor of Overexpression of CO 1 Negatively Regulates Dark-Induced Leaf Degreening and Senescence by Directly Repressing Pheophytinase and Other Senescence-Associated Genes in Arabidopsis.

    Science.gov (United States)

    Chen, Junyi; Zhu, Xiaoyu; Ren, Jun; Qiu, Kai; Li, Zhongpeng; Xie, Zuokun; Gao, Jiong; Zhou, Xin; Kuai, Benke

    2017-03-01

    Although the biochemical pathway of chlorophyll (Chl) degradation has been largely elucidated, how Chl is rapidly yet coordinately degraded during leaf senescence remains elusive. Pheophytinase (PPH) is the enzyme for catalyzing the removal of the phytol group from pheophytin a , and PPH expression is significantly induced during leaf senescence. To elucidate the transcriptional regulation of PPH , we used a yeast ( Saccharomyces cerevisiae ) one-hybrid system to screen for its trans-regulators. SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a key flowering pathway integrator, was initially identified as one of the putative trans-regulators of PPH After dark treatment, leaves of an SOC1 knockdown mutant ( soc1-6 ) showed an accelerated yellowing phenotype, whereas those of SOC1 -overexpressing lines exhibited a partial stay-green phenotype. SOC1 and PPH expression showed a negative correlation during leaf senescence. Substantially, SOC1 protein could bind specifically to the CArG box of the PPH promoter in vitro and in vivo, and overexpression of SOC1 significantly inhibited the transcriptional activity of the PPH promoter in Arabidopsis ( Arabidopsis thaliana ) protoplasts. Importantly, soc1-6 pph-1 (a PPH knockout mutant) double mutant displayed a stay-green phenotype similar to that of pph-1 during dark treatment. These results demonstrated that SOC1 inhibits Chl degradation via negatively regulating PPH expression. In addition, measurement of the Chl content and the maximum photochemical efficiency of photosystem II of soc1-6 and SOC1-OE leaves after dark treatment suggested that SOC1 also negatively regulates the general senescence process. Seven SENESCENCE-ASSOCIATED GENES ( SAGs ) were thereafter identified as its potential target genes, and NONYELLOWING1 and SAG113 were experimentally confirmed. Together, we reveal that SOC1 represses dark-induced leaf Chl degradation and senescence in general in Arabidopsis. © 2017 American Society of Plant Biologists. All

  19. A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition

    Science.gov (United States)

    Moutinho-Santos, Tatiana

    2013-01-01

    Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor. PMID:23609535

  20. Suppressor analysis reveals a role for SecY in the SecA2-dependent protein export pathway of Mycobacteria.

    Science.gov (United States)

    Ligon, Lauren S; Rigel, Nathan W; Romanchuk, Artur; Jones, Corbin D; Braunstein, Miriam

    2013-10-01

    All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.

  1. Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

    Science.gov (United States)

    Riquelme, Sebastián A; Hopkins, Benjamin D; Wolfe, Andrew L; DiMango, Emily; Kitur, Kipyegon; Parsons, Ramon; Prince, Alice

    2017-12-19

    The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl -/- mice, which lack the NH 2 -amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy. Published by Elsevier Inc.

  2. Registered report: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs.

    Science.gov (United States)

    Phelps, Mitch; Coss, Chris; Wang, Hongyan; Cook, Matthew

    2016-03-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Coding-Independent Regulation of the Tumor Suppressor PTEN by Competing Endogenous 'mRNAs' by Tay and colleagues, published in Cell in 2011 (Tay et al., 2011). The experiments to be replicated are those reported in Figures 3C, 3D, 3G, 3H, 5A and 5B, and in Supplemental Figures 3A and B. Tay and colleagues proposed a new regulatory mechanism based on competing endogenous RNAs (ceRNAs), which regulate target genes by competitive binding of shared microRNAs. They test their model by identifying and confirming ceRNAs that target PTEN. In Figure 3A and B, they report that perturbing expression of putative PTEN ceRNAs affects expression of PTEN. This effect is dependent on functional microRNA machinery (Figure 3G and H), and affects the pathway downstream of PTEN itself (Figures 5A and B). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

  3. The bifunctional abiotic stress signalling regulator and endogenous RNA silencing suppressor FIERY1 is required for lateral root formation

    KAUST Repository

    Chen, Hao

    2010-09-28

    The Arabidopsis FIERY1 (FRY1) locus was originally identified as a negative regulator of stress-responsive gene expression and later shown to be required for suppression of RNA silencing. In this study we discovered that the FRY1 locus also regulates lateral root formation. Compared with the wild type, fry1 mutant seedlings generated significantly fewer lateral roots under normal growth conditions and also exhibited a dramatically reduced sensitivity to auxin in inducing lateral root initiation. Using transgenic plants that overexpress a yeast homolog of FRY1 that possesses only the 3\\', 5\\'-bisphosphate nucleotidase activity but not the inositol 1-phosphatase activity, we demonstrated that the lateral root phenotypes in fry1 result from loss of the nucleotidase activity. Furthermore, a T-DNA insertion mutant of another RNA silencing suppressor, XRN4 (but not XRN2 or XRN3), which is an exoribonuclease that is inhibited by the substrate of the FRY1 3\\', 5\\'-bisphosphate nucleotidase, exhibits similar lateral root defects. Although fry1 and xrn4 exhibited reduced sensitivity to ethylene, our experiments demonstrated that restoration of ethylene sensitivity in the fry1 mutant is not sufficient to rescue the lateral root phenotypes of fry1. Our results indicate that RNA silencing modulated by FRY1 and XRN4 plays an important role in shaping root architecture. © 2010 Blackwell Publishing Ltd.

  4. Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

    DEFF Research Database (Denmark)

    Andresen, Christina Aaen; Smedegaard, Stine; Sylvestersen, Kathrine Beck

    2014-01-01

    The Ankyrin and SOCS (Suppressor of Cytokine Signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting with 18 members in humans, the identity of the physiological targets of the Asb protei...

  5. Linker Histone H1.2 Directs Genome-wide Chromatin Association of the Retinoblastoma Tumor Suppressor Protein and Facilitates Its Function

    Directory of Open Access Journals (Sweden)

    Shonagh Munro

    2017-06-01

    Full Text Available The retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest.

  6. The tumor suppressor phosphatase PP2A-B56α regulates stemness and promotes the initiation of malignancies in a novel murine model.

    Directory of Open Access Journals (Sweden)

    Mahnaz Janghorban

    Full Text Available Protein phosphatase 2A (PP2A is a ubiquitously expressed Serine-Threonine phosphatase mediating 30-50% of protein phosphatase activity. PP2A functions as a heterotrimeric complex, with the B subunits directing target specificity to regulate the activity of many key pathways that control cellular phenotypes. PP2A-B56α has been shown to play a tumor suppressor role and to negatively control c-MYC stability and activity. Loss of B56α promotes cellular transformation, likely at least in part through its regulation of c-MYC. Here we report generation of a B56α hypomorph mouse with very low B56α expression that we used to study the physiologic activity of the PP2A-B56α phosphatase. The predominant phenotype we observed in mice with B56α deficiency in the whole body was spontaneous skin lesion formation with hyperproliferation of the epidermis, hair follicles and sebaceous glands. Increased levels of c-MYC phosphorylation on Serine62 and c-MYC activity were observed in the skin lesions of the B56αhm/hm mice. B56α deficiency was found to increase the number of skin stem cells, and consistent with this, papilloma initiation was accelerated in a carcinogenesis model. Further analysis of additional tissues revealed increased inflammation in spleen, liver, lung, and intestinal lymph nodes as well as in the skin lesions, resembling elevated extramedullary hematopoiesis phenotypes in the B56αhm/hm mice. We also observed an increase in the clonogenicity of bone marrow stem cells in B56αhm/hm mice. Overall, this model suggests that B56α is important for stem cells to maintain homeostasis and that B56α loss leading to increased activity of important oncogenes, including c-MYC, can result in aberrant cell growth and increased stem cells that can contribute to the initiation of malignancy.

  7. Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-β signaling

    Directory of Open Access Journals (Sweden)

    Linehan W Marston

    2010-06-01

    Full Text Available Abstract Background Germline mutations in the FLCN gene are responsible for the development of fibrofolliculomas, lung cysts and renal neoplasia in Birt-Hogg-Dube' (BHD syndrome. The encoded protein folliculin (FLCN is conserved across species but contains no classic motifs or domains and its function remains unknown. Somatic mutations or loss of heterozygosity in the remaining wild type copy of the FLCN gene have been found in renal tumors from BHD patients suggesting that FLCN is a classic tumor suppressor gene. Results To examine the tumor suppressor function of FLCN, wild-type or mutant FLCN (H255R was stably expressed in a FLCN-null renal tumor cell line, UOK257, derived from a BHD patient. When these cells were injected into nude mice, tumor development was inversely dependent upon the level of wild-type FLCN expression. We identified genes that were differentially expressed in the cell lines with or without wild-type FLCN, many of which are involved in TGF-β signaling, including TGF-β2 (TGFB2, inhibin β A chain (INHBA, thrombospondin 1 (THBS1, gremlin (GREM1, and SMAD3. In support of the in vitro data, TGFB2, INHBA, THBS1 and SMAD3 expression levels were significantly lower in BHD-associated renal tumors compared with normal kidney tissue. Although receptor mediated SMAD phosphorylation was not affected, basal and maximal TGF-β-induced levels of TGFB2, INHBA and SMAD7 were dramatically reduced in FLCN-null cells compared with FLCN-restored cells. Secreted TGF-β2 and activin A (homo-dimer of INHBA protein levels were also lower in FLCN-null cells compared with FLCN-restored cells. Consistent with a growth suppressive function, activin A (but not TGF-β2 completely suppressed anchorage-independent growth of FLCN-null UOK257 cells. Conclusions Our data demonstrate a role for FLCN in the regulation of key molecules in TGF-β signaling and confirm deregulation of their expression in BHD-associated renal tumors. Thus, deregulation of genes

  8. Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21.

    LENUS (Irish Health Repository)

    Sheedy, FJ

    2009-11-29

    The tumor suppressor PDCD4 is a proinflammatory protein that promotes activation of the transcription factor NF-kappaB and suppresses interleukin 10 (IL-10). Here we found that mice deficient in PDCD4 were protected from lipopolysaccharide (LPS)-induced death. The induction of NF-kappaB and IL-6 by LPS required PDCD4, whereas LPS enhanced IL-10 induction in cells lacking PDCD4. Treatment of human peripheral blood mononuclear cells with LPS resulted in lower PDCD4 expression, which was due to induction of the microRNA miR-21 via the adaptor MyD88 and NF-kappaB. Transfection of cells with a miR-21 precursor blocked NF-kappaB activity and promoted IL-10 production in response to LPS, whereas transfection with antisense oligonucleotides to miR-21 or targeted protection of the miR-21 site in Pdcd4 mRNA had the opposite effect. Thus, miR-21 regulates PDCD4 expression after LPS stimulation.

  9. The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

    DEFF Research Database (Denmark)

    Jenal, Mathias; Trinh, Emmanuelle; Britschgi, Christian

    2009-01-01

    The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened...

  10. Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

    NARCIS (Netherlands)

    Ruane, Peter T; Gumy, Laura F|info:eu-repo/dai/nl/337608334; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502; Allan, Victoria J

    2016-01-01

    Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members,

  11. miR-203 Acts as a Tumor Suppressor Gene in Osteosarcoma by Regulating RAB22A.

    Directory of Open Access Journals (Sweden)

    Dawei Yang

    Full Text Available microRNAs (miRNAs, small noncoding RNAs of 19-25 nt, play an important roles in the pathological processes of tumorigenesis. The object of this study was to study the expression and function of miR-203 and to found its target gene in osteosarcoma. In our study, we found the expression level of miR-203 was significantly downregulated in osteosarcoma cell lines and tissues. In addition, overexpression of miR-203 inhibited the osteosarcoma cell proliferation and migration and inhibited Mesenchymal-to-Epithelial reversion Transition (MErT. Moreover, we identified RAB22A as a direct target of miR-203 and RAB22A overexpression blocks the roles of miR-203 in osteosarcoma cell. Furthermore, we demonstrated that RAB22A expression was upregulated in human osteosarcoma cell lines and tissues. Take together, our results demonstrated that miR-203 act as a tumor suppressor miRNA through regulating RAB22A expression and suggested its involvement in osteosarcoma progression and carcinogenesis.

  12. Regulation of the insulin-like developmental pathway of Caenorhabditis elegans by a homolog of the PTEN tumor suppressor gene.

    Science.gov (United States)

    Gil, E B; Malone Link, E; Liu, L X; Johnson, C D; Lees, J A

    1999-03-16

    The human PTEN tumor suppressor gene is mutated in a wide variety of sporadic tumors. To determine the function of PTEN in vivo we have studied a PTEN homolog in Caenorhabditis elegans. We have generated a strong loss-of-function allele of the PTEN homolog and shown that the deficient strain is unable to enter dauer diapause. An insulin-like phosphatidylinositol 3-OH kinase (PI3'K) signaling pathway regulates dauer-stage entry. Mutations in either the daf-2 insulin receptor-like (IRL) gene or the age-1 encoded PI3'K catalytic subunit homolog cause constitutive dauer formation and also affect the life span, brood size, and metabolism of nondauer animals. Strikingly, loss-of-function mutations in the age-1 PI3'K and daf-2 IRL genes are suppressed by loss-of-function mutations in the PTEN homolog. We establish that the PTEN homolog is encoded by daf-18, a previously uncloned gene that has been shown to interact genetically with the DAF-2 IRL AGE-1 PI3'K signaling pathway. This interaction provides clear genetic evidence that PTEN acts to antagonize PI3'K function in vivo. Given the conservation of the PI3'K signaling pathway between C. elegans and mammals, the analysis of daf-18 PTEN mutant nematodes should shed light on the role of human PTEN in the etiology of metabolic disease, aging, and cancer.

  13. Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules

    NARCIS (Netherlands)

    Schnettler, E.; Hemmes, J.C.; Huisman, R.; Goldbach, R.W.; Prins, M.W.; Kormelink, R.J.M.

    2010-01-01

    The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs

  14. E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer

    DEFF Research Database (Denmark)

    Lu, Z; Luo, R Z; Peng, H

    2006-01-01

    to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies...... and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3...... cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4...

  15. [Effects of moxibustion intervention on inflammatory reactions and expression of suppressor of cytokine signaling proteins of synovium cells in rheumatoid arthritis rabbits].

    Science.gov (United States)

    Yang, Xin; Liu, Xu-Guang; Wang, Yue; Yang, Shen-Qiao; Jin, Rong-Jiang

    2013-04-01

    down-regulating expression of SOCS 1 and SOCS 3 proteins by suppressing negative feedback regulatory JAK/STAT pathway in synovial cells. [KEY WORDS] Moxibustion; Rheumatoid arthritis; Inflammatory reactions; Synovial cells; Suppressor of cytokine signaling proteins; Negative-feedback regulatory factors

  16. ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor

    Science.gov (United States)

    Zeqiraj, Elton; Filippi, Beatrice Maria; Goldie, Simon; Navratilova, Iva; Boudeau, Jérôme; Deak, Maria; Alessi, Dario R.; van Aalten, Daan M. F.

    2009-01-01

    Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations. PMID:19513107

  17. ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor.

    Science.gov (United States)

    Zeqiraj, Elton; Filippi, Beatrice Maria; Goldie, Simon; Navratilova, Iva; Boudeau, Jérôme; Deak, Maria; Alessi, Dario R; van Aalten, Daan M F

    2009-06-09

    Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.

  18. ATP and MO25alpha regulate the conformational state of the STRADalpha pseudokinase and activation of the LKB1 tumour suppressor.

    Directory of Open Access Journals (Sweden)

    Elton Zeqiraj

    2009-06-01

    Full Text Available Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADalpha in complex with MO25alpha. The structure reveals an intricate web of interactions between STRADalpha and MO25alpha involving the alphaC-helix of STRADalpha, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADalpha binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADalpha for MO25alpha, and conversely, binding of MO25alpha promotes interaction of STRADalpha with ATP. Mutagenesis studies reveal that association of STRADalpha with either ATP or MO25alpha is essential for LKB1 activation. We conclude that ATP and MO25alpha cooperate to maintain STRADalpha in an "active" closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADalpha that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE syndrome. We demonstrate this mutation destabilizes STRADalpha and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADalpha to activate LKB1 is dependent on a closed "active" conformation, aided by ATP and MO25alpha binding. Thus, the function of STRADalpha is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.

  19. High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

    Science.gov (United States)

    Lösche, Matthias

    2012-02-01

    The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration PTEN's regulatory C-terminal tail is displaced from the membrane and

  20. INHIBITION OF THE DNA-BINDING ACTIVITY OF DROSOPHILA SUPPRESSOR OF HAIRLESS AND OF ITS HUMAN HOMOLOG, KBF2/RBP-J-KAPPA, BY DIRECT PROTEIN-PROTEIN INTERACTION WITH DROSOPHILA HAIRLESS

    NARCIS (Netherlands)

    BROU, C; LOGEAT, F; LECOURTOIS, M; VANDEKERCKHOVE, Joël; KOURILSKY, P; SCHWEISGUTH, F; ISRAEL, A

    1994-01-01

    We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed

  1. Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems.

    Directory of Open Access Journals (Sweden)

    Wenlin An

    Full Text Available Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI with a destabilizing domain (DD specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.

  2. Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

    Directory of Open Access Journals (Sweden)

    Bruno Pagano

    Full Text Available The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

  3. The Ebola virus VP35 protein is a suppressor of RNA silencing

    NARCIS (Netherlands)

    Haasnoot, J.; Vries, de W.; Geutjes, E.J.; Prins, M.W.; Haan, de P.; Berkhout, B.

    2007-01-01

    RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is

  4. Evidence for protein 4.1B acting as a metastasis suppressor

    Czech Academy of Sciences Publication Activity Database

    Cavanna, T.; Pokorná, Eva; Veselý, Pavel; Gray, C.; Zicha, D.

    2007-01-01

    Roč. 120, č. 4 (2007), s. 606-616 ISSN 0021-9533 Institutional research plan: CEZ:AV0Z50520514 Keywords : 4.1B protein * metastasis * migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.383, year: 2007

  5. PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

    Science.gov (United States)

    Makker, Annu; Goel, Madhu Mati; Mahdi, Abbas Ali; Bhatia, Vikram; Das, Vinita; Agarwal, Anjoo; Pandey, Amita

    2016-05-01

    Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (PPTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

  6. Autopalmitoylation of TEAD Proteins Regulates Transcriptional Output of Hippo Pathway

    Science.gov (United States)

    Chan, PuiYee; Han, Xiao; Zheng, Baohui; DeRan, Michael; Yu, Jianzhong; Jarugumilli, Gopala K.; Deng, Hua; Pan, Duojia; Luo, Xuelian; Wu, Xu

    2016-01-01

    TEA domain (TEAD) transcription factors bind to the co-activator YAP/TAZ, and regulate the transcriptional output of Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches fatty acid (palmitate) to cysteine residues, and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities, and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs, and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation is required for TEAD’s binding to YAP/TAZ, but dispensable for the binding to Vgll4 tumor suppressor. In addition, palmitoylation does not alter TEAD’s localization. Moreover, TEAD palmitoylation-deficient mutants impaired TAZ-mediated muscle differentiation in vitro, and Yorkie-mediated tissue overgrowth in Drosophila in vivo. Our study directly linked autopalmitoylation to the transcriptional regulation of Hippo pathway. PMID:26900866

  7. Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma

    OpenAIRE

    Marko, Tracy A.; Shamsan, Ghaidan A.; Edwards, Elizabeth N.; Hazelton, Paige E.; Rathe, Susan K.; Cornax, Ingrid; Overn, Paula R.; Varshney, Jyotika; Diessner, Brandon J.; Moriarity, Branden S.; O?Sullivan, M. Gerard; Odde, David J.; Largaespada, David A.

    2016-01-01

    Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 n...

  8. Mast cells down-regulate CD4+CD25+ T regulatory cell suppressor function via histamine H1 receptor interaction.

    Science.gov (United States)

    Forward, Nicholas A; Furlong, Suzanne J; Yang, Yongjun; Lin, Tong-Jun; Hoskin, David W

    2009-09-01

    Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (T(reg)) cell function. In this study, we show for the first time that the capacity of murine CD4(+)CD25(+) T(reg) cells to suppress in vitro proliferation by CD4(+)CD25(-) T responder (T(resp)) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcepsilonR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited T(reg) cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored T(reg) cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of T(reg) cells with loratadine, but not famotidine, rescued T(reg) cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited T(reg) cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both T(reg) cells and T(resp) cells expressed H1 receptors. Exposure to histamine caused T(reg) cells to express lower levels of CD25 and the T(reg) cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited T(reg) cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in T(reg) cell suppressor function, thereby enhancing the development of protective immunity.

  9. Identification of a genetic interaction between the tumor suppressor EAF2 and the retinoblastoma protein (Rb) signaling pathway in C. elegans and prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Liquan; Wang, Dan [Department of Urology, The University of Pittsburgh, 5200 Centre Avenue, Pittsburgh, PA 15216 (United States); Fisher, Alfred L., E-mail: fishera2@uthscsa.edu [Division of Geriatrics, Gerontology, and Palliative Medicine, Department of Medicine, UTHSCSA, San Antonio, TX 78229 (United States); Center for Healthy Aging, UTHSCSA, San Antonio, TX 78229 (United States); GRECC, STVAHCS, San Antonio, TX 78229 (United States); Wang, Zhou, E-mail: wangz2@upmc.edu [Department of Urology, The University of Pittsburgh, 5200 Centre Avenue, Pittsburgh, PA 15216 (United States); GRECC, STVAHCS, San Antonio, TX 78229 (United States)

    2014-05-02

    Highlights: • RNAi screen identified genetic enhancers for the C. elegans homolog of EAF2. • EAF2 and RBBP4 proteins physically bind to each other and alter transcription. • Overexpression of EAF2 and RBBP4 induces the cell death in prostate cancer cells. - Abstract: The tumor suppressor EAF2 is regulated by androgen signaling and associated with prostate cancer. While EAF2 and its partner ELL have been shown to be members of protein complexes involved in RNA polymerase II transcriptional elongation, the biologic roles for EAF2 especially with regards to the development of cancer remains poorly understood. We have previously identified the eaf-1 gene in Caenorhabditiselegans as the ortholog of EAF2, and shown that eaf-1 interacts with the ELL ortholog ell-1 to control development and fertility in worms. To identify genetic pathways that interact with eaf-1, we screened RNAi libraries consisting of transcription factors, phosphatases, and chromatin-modifying factors to identify genes which enhance the effects of eaf-1(tm3976) on fertility. From this screen, we identified lin-53, hmg-1.2, pha-4, ruvb-2 and set-6 as hits. LIN-53 is the C. elegans ortholog of human retinoblastoma binding protein 4/7 (RBBP 4/7), which binds to the retinoblastoma protein and inhibits the Ras signaling pathway. We find that lin-53 showed a synthetic interaction with eaf-1(tm3976) where knockdown of lin-53 in an eaf-1(tm3976) mutant resulted in sterile worms. This phenotype may be due to cell death as the treated worms contain degenerated embryos with increased expression of the ced-1:GFP cell death marker. Further we find that the interaction between eaf-1 and lin-53/RBBP4/7 also exists in vertebrates, which is reflected by the formation of a protein complex between EAF2 and RBBP4/7. Finally, overexpression of either human EAF2 or RBBP4 in LNCaP cells induced the cell death while knockdown of EAF2 in LNCaP enhanced cell proliferation, indicating an important role of EAF2 in

  10. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    International Nuclear Information System (INIS)

    Machowska, Magdalena; Wachowicz, Katarzyna; Sopel, Mirosław; Rzepecki, Ryszard

    2014-01-01

    Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti-proliferative effect of nuclear

  11. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    Science.gov (United States)

    2014-01-01

    Background Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Methods Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. Results We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti

  12. Generation of protein-specific and alloantigen-specific suppressor cells following total lymphoid irradiation in mice

    International Nuclear Information System (INIS)

    Slavin, S.; Zan-Bar, I.; Strober, S.

    1979-01-01

    The presence of donor-type specific suppressor cells was demonstrated in C57BL/Ka → BALB/c BM chimeras in both in vivo and in vitro experiments. In both experiments tolerance to either BSA of C57BL/Ka tissue antigens could be transferred into adoptive recipients. In view of the data obtained in BSA-tolerant mice, it is likely that the suppressor cells in the chimeras were also T cells in origin; however, formal proof has yet to be obtained. We conclude that antigen-specific suppressor cells are generated following TLI. Specific transplantation tolerance obtained by immunomanipulation rather than by prolonged use of nonspecific immunosuppressive agents is the goal of clinical BM and organ transplantation. Due to the experience accumulated in patients with Hodgkin's disease regarding the effects and relative safety of using TLI, it may soon become a new clinical tool for BM and organ transplantation in man

  13. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    International Nuclear Information System (INIS)

    Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

    2014-01-01

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept

  14. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    Energy Technology Data Exchange (ETDEWEB)

    Reidick, Christina [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany); El Magraoui, Fouzi; Meyer, Helmut E. [Biomedical Research, Human Brain Proteomics II, Leibniz-Institut für Analytische Wissenschaften-ISAS, Dortmund 44139 (Germany); Stenmark, Harald [Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo 0310 (Norway); Platta, Harald W., E-mail: harald.platta@rub.de [Biochemie Intrazellulärer Transportprozesse, Ruhr-Universität Bochum, Bochum 44801 (Germany)

    2014-12-23

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

  15. Polyglutamine-rich suppressors of huntingtin toxicity act upstream of Hsp70 and Sti1 in spatial quality control of amyloid-like proteins.

    Directory of Open Access Journals (Sweden)

    Katie J Wolfe

    Full Text Available Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS. High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic

  16. P53 tumor suppressor gene and protein expression is altered in cell lines derived from spontaneous and alpha-radiation-induced canine lung tumors

    International Nuclear Information System (INIS)

    Tierney, L.A.; Johnson, N.F.; Lechner, J.F.

    1994-01-01

    Mutations in the p53 tumor suppressor gene are the most frequently occurring gene alterations in malignant human cancers, including lung cancer. In lung cancer, common point mutations within conserved exons of the p53 gene result in a stabilized form of mutant protein which is detectable in most cases by immunohistochemistry. In addition to point mutations, allelic loss, rearrangements, and deletions of the p53 gene have also been detected in both human and rodent tumors. It has been suggested that for at least some epithelial neoplasms, the loss of expression of wild-type p53 protein may be more important for malignant transformation than the acquisition of activating mutations. Mechanisms responsible for the loss of expression of wild-type protein include gene deletion or rearrangement, nonsense or stop mutations, mutations within introns or upstream regulatory regions of the gene, and accelerated rates of degradation of the protein by DNA viral oncoproteins

  17. Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells.

    Science.gov (United States)

    Kim, Myeong-Ok; Choe, Min Ho; Yoon, Yi Na; Ahn, Jiyeon; Yoo, Minjin; Jung, Kwan-Young; An, Sungkwan; Hwang, Sang-Gu; Oh, Jeong Su; Kim, Jae-Sung

    2017-11-15

    Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged asan anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide asa CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide asa PP2A-activating drug in the clinical treatment of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    Directory of Open Access Journals (Sweden)

    Bianco Linda

    2009-11-01

    Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

  19. Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor

    OpenAIRE

    Liu, Xin; Marmorstein, Ronen

    2007-01-01

    The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transac...

  20. A label-free electrochemical test for DNA-binding activities of tumor suppressor protein p53 using immunoprecipitation at magnetic beads

    Czech Academy of Sciences Publication Activity Database

    Němcová, Kateřina; Havran, Luděk; Šebest, Peter; Brázdová, Marie; Pivoňková, Hana; Fojta, Miroslav

    2010-01-01

    Roč. 668, č. 2 (2010), s. 166-170 ISSN 0003-2670 R&D Projects: GA AV ČR(CZ) IAA500040701; GA ČR(CZ) GP204/07/P476; GA ČR(CZ) GA204/08/1560; GA AV ČR(CZ) 1QS500040581; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : electrochemical analysis * label-free detection * tumor suppressor protein p53 Subject RIV: BO - Biophysics Impact factor: 4.310, year: 2010

  1. Foxa2, a novel protein partner of the tumour suppressor menin, is deregulated in mouse and human MEN1 glucagonomas.

    Science.gov (United States)

    Bonnavion, Rémy; Teinturier, Romain; Gherardi, Samuele; Leteurtre, Emmanuelle; Yu, Run; Cordier-Bussat, Martine; Du, Rui; Pattou, François; Vantyghem, Marie-Christine; Bertolino, Philippe; Lu, Jieli; Zhang, Chang Xian

    2017-05-01

    Foxa2, known as one of the pioneer factors, plays a crucial role in islet development and endocrine functions. Its expression and biological functions are regulated by various factors, including, in particular, insulin and glucagon. However, its expression and biological role in adult pancreatic α-cells remain elusive. In the current study, we showed that Foxa2 was overexpressed in islets from α-cell-specific Men1 mutant mice, at both the transcriptional level and the protein level. More importantly, immunostaining analyses showed its prominent nuclear accumulation, specifically in α-cells, at a very early stage after Men1 disruption. Similar nuclear FOXA2 expression was also detected in a substantial proportion (12/19) of human multiple endocrine neoplasia type 1 (MEN1) glucagonomas. Interestingly, our data revealed an interaction between Foxa2 and menin encoded by the Men1 gene. Furthermore, using several approaches, we demonstrated the relevance of this interaction in the regulation of two tested Foxa2 target genes, including the autoregulation of the Foxa2 promoter by Foxa2 itself. The current study establishes menin, a novel protein partner of Foxa2, as a regulator of Foxa2, the biological functions of which extend beyond the pancreatic endocrine cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  2. BAR domain proteins regulate Rho GTPase signaling.

    Science.gov (United States)

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

  3. Modulator of Apoptosis 1: A Highly Regulated RASSF1A-Interacting BH3-Like Protein

    OpenAIRE

    Law, Jennifer; Yu, Victor C.; Baksh, Shairaz

    2012-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in both the intrinsic and extrinsic modes of cell death or apoptosis. MOAP-1 is part of the Ras association domain family 1A (RASSF1A)/MOAP-1 pro-apoptotic extrinsic signaling pathway that regulates apoptosis by utilizing death receptors such as tumor necrosis factor α (TNF α ) or TNF-related apoptosis-inducing ligand (TRAIL) to inhibit abnormal growth. RASSF1A is a bona fide tumor suppressor gene that is epigenetica...

  4. Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

    Science.gov (United States)

    Lubecka-Pietruszewska, Katarzyna; Kaufman-Szymczyk, Agnieszka; Stefanska, Barbara; Cebula-Obrzut, Barbara; Smolewski, Piotr; Fabianowska-Majewska, Krystyna

    2015-01-01

    Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1 , p53 , and p21 , in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. In both MCF-7 and MDA-MB-231 cells, SFN at IC 50 (22 and 46 μ M , respectively) and a physiologically relevant 10 μ M concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene upregulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

  5. Regulation of protein turnover by heat shock proteins.

    Science.gov (United States)

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14ARF tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Dimri, Goberdhan P.; Itahana, Koji; Acosta, Meileen; Campisi, Judith

    1999-11-05

    Normal cells do not divide indefinitely due to a process known as replicative senescence. Human cells arrest growth with a senescent phenotype when they acquire one or more critically short telomere as a consequence of cell division. Recent evidence suggests that certain types of DNA damage, chromatin remodeling, or oncogenic forms of Rasor Raf can also elicit a senescence response. We show here that E2F1, a multifunctional transcription factor that binds the retinoblastoma (pRb) tumor suppressor and can either promote or suppress tumorigenesis, induces a senescent phenotype when overexpressed in normal human fibroblasts. Normal human cells stably arrested proliferation and expressed several markers of replicative senescence in response to E2F1. This activity of E2F1 was independent of its pRb binding activity, but dependent on its ability to stimulate gene expression. The E2F1 target gene critical for the senescence response appeared to be the p14ARF tumor suppressor. Replicatively senescent human fibroblasts overexpressed p14ARF, and ectopic expression of p14ARF in presenescent cells induced a phenotype similar to that induced by E2F1. Consistent with a critical role for p14ARF, cells with compromised p53 function were immune to senescence induction by E2F1, as were cells deficient in p14ARF. Our findings support the idea that the senescence response is a critical tumor suppressive mechanism, provide an explanation for the apparently paradoxical roles of E2F1 in oncogenesis, and identify p14ARF as a potentially important mediator of the senescent phenotype.

  7. Runx proteins regulate Foxp3 expression

    OpenAIRE

    Bruno, Ludovica; Mazzarella, Luca; Hoogenkamp, Maarten; Hertweck, Arnulf; Cobb, Bradley S.; Sauer, Stephan; Hadjur, Suzana; Leleu, Marion; Naoe, Yoshinori; Telfer, Janice C.; Bonifer, Constanze; Taniuchi, Ichiro; Fisher, Amanda G.; Merkenschlager, Matthias

    2009-01-01

    Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regu...

  8. Membrane association of the PTEN tumor suppressor: neutron scattering and MD simulations reveal the structure of protein-membrane complexes.

    Science.gov (United States)

    Nanda, Hirsh; Heinrich, Frank; Lösche, Mathias

    2015-05-01

    Neutron reflection (NR) from planar interfaces is an emerging technology that provides unique and otherwise inaccessible structural information on disordered molecular systems such as membrane proteins associated with fluid bilayers, thus addressing one of the remaining challenges of structural biology. Although intrinsically a low-resolution technique, using structural information from crystallography or NMR allows the construction of NR models that describe the architecture of protein-membrane complexes at high resolution. In addition, a combination of these methods with molecular dynamics (MD) simulations has the potential to reveal the dynamics of protein interactions with the bilayer in atomistic detail. We review recent advances in this area by discussing the application of these techniques to the complex formed by the PTEN phosphatase with the plasma membrane. These studies provide insights in the cellular regulation of PTEN, its interaction with PI(4,5)P2 in the inner plasma membrane and the pathway by which its substrate, PI(3,4,5)P3, accesses the PTEN catalytic site. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Ric-8 regulation of heterotrimeric G proteins.

    Science.gov (United States)

    Tall, Gregory G

    2013-06-01

    Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.

  10. Presenilin-2 regulates the degradation of RBP-Jk protein through p38 mitogen-activated protein kinase.

    Science.gov (United States)

    Kim, Su-Man; Kim, Mi-Yeon; Ann, Eun-Jung; Mo, Jung-Soon; Yoon, Ji-Hye; Park, Hee-Sae

    2012-03-01

    Transcriptional regulation performs a central role in Notch1 signaling by recombining binding protein Suppressor of Hairless (RBP-Jk)--a signaling pathway that is widely involved in determination of cell fate. Our earlier work demonstrated the possible regulation of the Notch1-RBP-Jk pathway through protein degradation of RBP-Jk; however, the potential regulator for the degradation of RBP-Jk remains to be determined. Here, we report that the expression of endogenous and exogenous RBP-Jk was increased significantly in cells treated with proteasome- and lysosome-specific inhibitors. The effects of these inhibitors on RBP-Jk occurred in a dose- and time-dependent manner. The level of RBP-Jk protein was higher in presenilin-2 (PS2)-knockout cells than in presenilin-1 (PS1)-knockout cells. Furthermore, the level of RBP-Jk was decreased by expression of PS2 in PS1 and PS2 double-knockout cells. We also found that PS1-knockout cells treated with a specific inhibitor of p38 mitogen-activated protein kinase ∂ (MAPK) had significantly increased levels of RBP-Jk. p38 MAPK phosphorylates RBP-Jk at Thr339 by physical binding, which subsequently induces the degradation and ubiquitylation of the RBP-Jk protein. Collectively, our results indicate that PS2 modulates the degradation of RBP-Jk through phosphorylation by p38 MAPK.

  11. The protein phosphatase activity of PTEN is essential for regulating neural stem cell differentiation.

    Science.gov (United States)

    Lyu, Jingwen; Yu, Xiuya; He, Lingjie; Cheng, Tianlin; Zhou, Jingjing; Cheng, Cheng; Chen, Zhifang; Cheng, Guoqiang; Qiu, Zilong; Zhou, Wenhao

    2015-04-18

    The tumor suppressor gene Phosphatase and tensin homolog (PTEN) is highly expressed in neural progenitor cells (NPCs) and plays an important role in development of the central nervous system. As a dual-specificity phosphatase, the loss of PTEN phosphatase activity has been linked to various diseases. Here we report that the protein phosphatase activity of Pten is critical for regulating differentiation of neural progenitor cells. First we found that deletion of Pten promotes neuronal differentiation. To determine whether the protein or lipid phosphatase activity is required for regulating neuronal differentiation, we generated phosphatase domain-specific Pten mutations. Interestingly, only expression of protein phosphatase-deficient mutant Y138L could mimic the effect of knocking down Pten, suggesting the protein phosphatase of Pten is critical for regulating NPC differentiation. Importantly, we showed that the wild-type and lipid phosphatase mutant (G129E) forms of Pten are able to rescue neuronal differentiation in Pten knockout NPCs, but mutants containing protein phosphatase mutant cannot. We further found that Pten-dependent dephosphorylation of CREB is critical for neuronal differentiation. Our data indicate that the protein phosphatase activity of PTEN is critical for regulating differentiation of NSCs during cortical development.

  12. Redox regulation of protein damage in plasma

    Directory of Open Access Journals (Sweden)

    Helen R. Griffiths

    2014-01-01

    In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites.

  13. Genetic modelling of PIM proteins in cancer: proviral tagging, cooperation with oncogenes, tumor suppressor genes and carcinogens.

    Directory of Open Access Journals (Sweden)

    Enara eAguirre

    2014-05-01

    Full Text Available The PIM proteins, which were initially discovered as proviral insertion sites in Moloney murine leukemia virus infection, are a family of highly homologous serine/threonine kinases that have been reported to be overexpressed in hematological malignancies and solid tumors. The PIM proteins have also been associated with metastasis and overall treatment responses and implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anticancer drug discovery. The use of retroviral insertional mutagenesis and refined approaches such as complementation tagging has allowed the identification of myc, pim and a third group of genes (including bmi1 and gfi1 as complementing genes in lymphomagenesis. Moreover, mouse modeling of human cancer has provided an understanding of the molecular pathways that are involved in tumor initiation and progression at the physiological level. In particular, genetically modified mice have allowed researchers to further elucidate the role of each of the Pim isoforms in various tumor types. PIM kinases have been identified as weak oncogenes because experimental overexpression in lymphoid tissue, prostate and liver induces tumors at a relatively low incidence and with a long latency. However, very strong synergistic tumorigenicity between Pim1/2 and c-Myc and other oncogenes has been observed in lymphoid tissues. Mouse models have also been used to study whether the inhibition of specific PIM isoforms is required to prevent carcinogen-induced sarcomas, indicating that the absence of Pim2 and Pim3 greatly reduces sarcoma growth and bone invasion; the extent of this effect is similar to that observed in the absence of all 3 isoforms. This review will summarize some of the animal models that have been used to understand the isoform-specific contribution of PIM kinases to tumorigenesis.

  14. DENN Domain Proteins: Regulators of Rab GTPases*

    Science.gov (United States)

    Marat, Andrea L.; Dokainish, Hatem; McPherson, Peter S.

    2011-01-01

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways. PMID:21330364

  15. DENN domain proteins: regulators of Rab GTPases.

    Science.gov (United States)

    Marat, Andrea L; Dokainish, Hatem; McPherson, Peter S

    2011-04-22

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.

  16. Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling

    Science.gov (United States)

    O'Day, Elizabeth; Li, Xiao Ling; Concepcion, Carla; Han, Yoon-Chi; Thiery, Jerome; Rajani, Danielle K.; Deutsch, Aaron; Hofmann, Oliver; Ventura, Andrea; Hide, Winston; Lieberman, Judy

    2011-01-01

    A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division. PMID:22102825

  17. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

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

    Science.gov (United States)

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

    2012-11-01

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

  19. Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor.

    Science.gov (United States)

    Liu, Xin; Marmorstein, Ronen

    2007-11-01

    The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.

  20. Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin; Marmorstein, Ronen (UPENN)

    2008-04-02

    The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.

  1. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Wei-Ru Huang

    Full Text Available Avian reovirus (ARV protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128 of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

  2. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

    Science.gov (United States)

    Huang, Wei-Ru; Chiu, Hung-Chuan; Liao, Tsai-Ling; Chuang, Kuo-Pin; Shih, Wing-Ling; Liu, Hung-Jen

    2015-01-01

    Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

  3. miR-339-5p regulates the p53 tumor-suppressor pathway by targeting MDM2

    DEFF Research Database (Denmark)

    Jansson, M D; Djodji Damas, Nkerorema; Lees, M

    2014-01-01

    proliferation in response to stress and represents the most commonly lost and mutated gene in human cancers. The function of p53 is inhibited by the MDM2 oncoprotein. Using a high-throughput screening approach, we identified miR-339-5p as a regulator of the p53 pathway. We demonstrate that this regulation...

  4. Tumor suppressors: enhancers or suppressors of regeneration?

    Science.gov (United States)

    Pomerantz, Jason H.; Blau, Helen M.

    2013-01-01

    Tumor suppressors are so named because cancers occur in their absence, but these genes also have important functions in development, metabolism and tissue homeostasis. Here, we discuss known and potential functions of tumor suppressor genes during tissue regeneration, focusing on the evolutionarily conserved tumor suppressors pRb1, p53, Pten and Hippo. We propose that their activity is essential for tissue regeneration. This is in contrast to suggestions that tumor suppression is a trade-off for regenerative capacity. We also hypothesize that certain aspects of tumor suppressor pathways inhibit regenerative processes in mammals, and that transient targeted modification of these pathways could be fruitfully exploited to enhance processes that are important to regenerative medicine. PMID:23715544

  5. Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

    Science.gov (United States)

    Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

    2017-08-15

    Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

  6. SOCS Proteins as Regulators of Inflammatory Responses Induced by Bacterial Infections: A Review

    Directory of Open Access Journals (Sweden)

    Skyla A. Duncan

    2017-12-01

    Full Text Available Severe bacterial infections can lead to both acute and chronic inflammatory conditions. Innate immunity is the first defense mechanism employed against invading bacterial pathogens through the recognition of conserved molecular patterns on bacteria by pattern recognition receptors (PRRs, especially the toll-like receptors (TLRs. TLRs recognize distinct pathogen-associated molecular patterns (PAMPs that play a critical role in innate immune responses by inducing the expression of several inflammatory genes. Thus, activation of immune cells is regulated by cytokines that use the Janus kinase/signal transducers and activators of transcription (JAK/STAT signaling pathway and microbial recognition by TLRs. This system is tightly controlled by various endogenous molecules to allow for an appropriately regulated and safe host immune response to infections. Suppressor of cytokine signaling (SOCS family of proteins is one of the central regulators of microbial pathogen-induced signaling of cytokines, principally through the inhibition of the activation of JAK/STAT signaling cascades. This review provides recent knowledge regarding the role of SOCS proteins during bacterial infections, with an emphasis on the mechanisms involved in their induction and regulation of antibacterial immune responses. Furthermore, the implication of SOCS proteins in diverse processes of bacteria to escape host defenses and in the outcome of bacterial infections are discussed, as well as the possibilities offered by these proteins for future targeted antimicrobial therapies.

  7. Merlin inhibits growth hormone-regulated Raf-ERKs pathways by binding to Grb2 protein

    International Nuclear Information System (INIS)

    Lim, Jung Yeon; Kim, Hongtae; Jeun, Sin-Soo; Kang, Seok-Gu; Lee, Kyung-Jin

    2006-01-01

    Numerous studies have suggested that the NF2 protein merlin is involved in the regulation of abnormal cell growth and proliferation. In this study, to better understand the merlin's mechanisms that contribute to the inhibition of tumorigenesis, we examined the potential action of merlin on the cell proliferative signaling pathways in response to growth hormone (GH). Merlin effectively attenuated the GH-induced serum response element (SRE) and Elk-1-mediated transcriptional activation, as well as the endogenous SRE-regulated gene c-fos expression in NIH3T3 cells. In addition, merlin prevented the Raf-1 complex activation process, which resulted in the suppression of MAP kinase/ERK, extracellular signal-regulated kinase (ERKs), and Elk-1 phosphorylation, which are the downstream signals of Raf-1. Moreover, it was shown that merlin interacted with endogenous growth factor receptor bound 2 (Grb2) protein and inhibited its expression. These results suggest that merlin contributes, via its protein-to-protein interaction with Grb2 and consequent inhibition of the MAPK pathways, to the regulation of the abnormal cell proliferation, and this provides a further mechanism underlying the tumor suppressor function of merlin

  8. The metastasis suppressor KISS1 is an intrinsically disordered protein slightly more extended than a random coil.

    Science.gov (United States)

    Ibáñez de Opakua, Alain; Merino, Nekane; Villate, Maider; Cordeiro, Tiago N; Ormaza, Georgina; Sánchez-Carbayo, Marta; Diercks, Tammo; Bernadó, Pau; Blanco, Francisco J

    2017-01-01

    The metastasis suppressor KISS1 is reported to be involved in the progression of several solid neoplasias, making it a promising molecular target for controlling their metastasis. The KISS1 sequence contains an N-terminal secretion signal and several dibasic sequences that are proposed to be the proteolytic cleavage sites. We present the first structural characterization of KISS1 by circular dichroism, multi-angle light scattering, small angle X-Ray scattering and NMR spectroscopy. An analysis of the KISS1 backbone NMR chemical shifts does not reveal any preferential conformation and deviation from a random coil ensemble. The backbone 15N transverse relaxation times indicate a mildly reduced mobility for two regions that are rich in bulky residues. The small angle X-ray scattering curve of KISS1 is likewise consistent with a predominantly random coil ensemble, although an ensemble optimization analysis indicates some preference for more extended conformations possibly due to positive charge repulsion between the abundant basic residues. Our results support the hypothesis that KISS1 mostly samples a random coil conformational space, which is consistent with its high susceptibility to proteolysis and the generation of Kisspeptin fragments.

  9. Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer.

    Science.gov (United States)

    Wen, Qirong; Liu, Yan; Lyu, Huabing; Xu, Xiaying; Wu, Qingxia; Liu, Ni; Yin, Qi; Li, Juan; Sheng, Xiujie

    2017-07-01

    The aims of this study were to investigate the functions of GAS5 as a tumor suppressor in cervical cancer and explore the mechanism. The expression of GAS5 and microRNA 21 (miR-21) was detected in primary cervical cancer tissue specimens, as well as in cervical cancer cell lines. We identified the interaction of GAS5 and miR-21 by quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter assay. We also studied the functions of GAS5 in proliferation, apoptosis, migration, and invasion in cervical cancer cells in vitro and vivo. Finally, the impact of GAS5 on cisplatin resistance and its mechanism in cervical cancer cells was also identified. The expression of GAS5 and miR-21 was detected in primary cervical cancer tissue specimens, as well as in cervical cancer cell lines. GAS5, which is a tumor suppressor playing roles in inhibiting the malignancy of cervical cancer cells, including proliferation in vivo and vitro, migration, and invasion, has a low expression in cervical cancer tissue and cervical cancer cell lines, whereas miR-21 expression is high. GAS5 significantly decreased the expression of miR-21, and there is a reciprocal repression of gene expression between GAS5 and miR-21. Besides, most importantly, we found that high expression of GAS5 and low expression of miR-21 can enhance the sensitivity of SiHa/cDDP cancer cells to cisplatin. A further experiment for identifying the mechanism of cisplatin resistance by GAS5 showed that GAS5 can not only regulate phosphatase and tensin homolog through miR-21 but also influence the phosphorylation of Akt. Our results indicate that GAS5 is a direct target of miR-21 and can predict the clinical staging of cervical cancer. Most importantly, GAS5 can also influence cisplatin resistance in cervical cancer via regulating the phosphorylation of Akt. All of these suggest that GAS5 may be a novel therapeutic target for treating cervical cancer.

  10. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  11. Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

    International Nuclear Information System (INIS)

    Davis, Sally J; Choong, David YH; Ramakrishna, Manasa; Ryland, Georgina L; Campbell, Ian G; Gorringe, Kylie L

    2011-01-01

    MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort

  12. The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

    LENUS (Irish Health Repository)

    Sernbo, Sandra

    2011-09-24

    Abstract Background The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking. Methods SOX11 expression and clinicopathological data was compared using χ2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation. Results SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5\\'-Aza-2\\'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11. Conclusions SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours.

  13. Mesenchymal Stem Cells (MSC Regulate Activation of Granulocyte-Like Myeloid Derived Suppressor Cells (G-MDSC in Chronic Myeloid Leukemia Patients.

    Directory of Open Access Journals (Sweden)

    Cesarina Giallongo

    Full Text Available It is well known that mesenchymal stem cells (MSC have a role in promotion of tumor growth, survival and drug-resistance in chronic myeloid leukemia (CML. Recent reports indicated that a subpopulation of myeloid cells, defined as granulocyte-like myeloid-derived suppressor cells (G-MDSC is increased in these patients. So far, the role of MSC in MDSC expansion and activation into the BM microenvironment remains unexplored. To address this question, here we use a specific experimental model in vitro, co-culturing MSC with peripheral blood mononucleated cells (PBMC from normal individuals, in order to generate MSC-educated G-MDSC. Although MSC of healthy donors (HD and CML patients were able to generate the same amount of MDSC, only CML-MSC-educated G-MDSC exhibited suppressive ability on autologous T lymphocytes. In addition, compared with HD-MSC, CML-MSC over-expressed some immunomodulatory factors including TGFβ, IL6 and IL10, that could be involved in MDSC activation. CML-MSC-educated G-MDSC expressed higher levels of ARG1, TNFα, IL1β, COX2 and IL6 than G-MDSC isolated from co-culture with HD-MSC. Our data provide evidence that CML-MSC may play a critical role in tumor microenvironment by orchestrating G-MDSC activation and regulating T lymphocytes-mediated leukemia surveillance, thus contributing to CML immune escape.

  14. Mesenchymal Stem Cells (MSC) Regulate Activation of Granulocyte-Like Myeloid Derived Suppressor Cells (G-MDSC) in Chronic Myeloid Leukemia Patients.

    Science.gov (United States)

    Giallongo, Cesarina; Romano, Alessandra; Parrinello, Nunziatina Laura; La Cava, Piera; Brundo, Maria Violetta; Bramanti, Vincenzo; Stagno, Fabio; Vigneri, Paolo; Chiarenza, Annalisa; Palumbo, Giuseppe Alberto; Tibullo, Daniele; Di Raimondo, Francesco

    2016-01-01

    It is well known that mesenchymal stem cells (MSC) have a role in promotion of tumor growth, survival and drug-resistance in chronic myeloid leukemia (CML). Recent reports indicated that a subpopulation of myeloid cells, defined as granulocyte-like myeloid-derived suppressor cells (G-MDSC) is increased in these patients. So far, the role of MSC in MDSC expansion and activation into the BM microenvironment remains unexplored. To address this question, here we use a specific experimental model in vitro, co-culturing MSC with peripheral blood mononucleated cells (PBMC) from normal individuals, in order to generate MSC-educated G-MDSC. Although MSC of healthy donors (HD) and CML patients were able to generate the same amount of MDSC, only CML-MSC-educated G-MDSC exhibited suppressive ability on autologous T lymphocytes. In addition, compared with HD-MSC, CML-MSC over-expressed some immunomodulatory factors including TGFβ, IL6 and IL10, that could be involved in MDSC activation. CML-MSC-educated G-MDSC expressed higher levels of ARG1, TNFα, IL1β, COX2 and IL6 than G-MDSC isolated from co-culture with HD-MSC. Our data provide evidence that CML-MSC may play a critical role in tumor microenvironment by orchestrating G-MDSC activation and regulating T lymphocytes-mediated leukemia surveillance, thus contributing to CML immune escape.

  15. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  16. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  17. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer

    Directory of Open Access Journals (Sweden)

    Joshua J. Thompson

    2018-02-01

    Full Text Available Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A is a heterotrimeric serine-threonine phosphatase—composed of a structural, regulatory, and catalytic subunit—that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components—especially the multitude of regulatory subunits—has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  18. Ubiquitin/SUMO modification regulates VHL protein stability and nucleocytoplasmic localization.

    Directory of Open Access Journals (Sweden)

    Qiliang Cai

    Full Text Available Functional inactivation of the von Hippel-Lindau (VHL tumor suppressor protein is linked to the development of several forms of cancer as well as oncogenic progression like sporadic renal clear-cell carcinomas (RCC. Despite the critical role played by VHL in destruction of hypoxia-inducible factor α (HIFα via ubiquitin-mediated proteolysis, very little is known about the post-translational modification which regulates VHL activity. Our previous study showed that the SUMO E3 ligase PIASy interacts with VHL and induces VHL SUMOylation on lysine residue 171 (Cai et al, PLoS ONE, 2010, 5(3:e9720. Here we further report that VHL also undergoes ubiquitylation on both lysine residues 171 and 196, which is blocked by PIASy. Moreover, using a VHL-SUMO1 or ubiquitin fusion protein, we found that ubiquitylated VHL is localized predominantly in the cytoplasm, while SUMOylated VHL results in increased VHL protein stability and nuclear redistribution. Interestingly, substitution of lysine 171 and 196 to arginine of VHL abrogates its inhibitory function on the transcriptional activity of HIFα, and tube formation in vitro. This demonstrates that post-translational modifications like ubiquitylation and SUMOylation contributes to VHL protein stability and nucleocytoplasmic shuttling, and that the overall function of VHL in tumor suppression may require a precise and dynamically regulated process which involves protein modification.

  19. Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders

    Directory of Open Access Journals (Sweden)

    Derek W. Stouth

    2017-11-01

    Full Text Available Protein arginine methyltransferases (PRMTs are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD. PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD, spinal muscular atrophy (SMA, and amyotrophic lateral sclerosis (ALS suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs. This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research.

  20. Protein trafficking and maturation regulate intramembrane proteolysis.

    Science.gov (United States)

    Morohashi, Yuichi; Tomita, Taisuke

    2013-12-01

    Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. A genome-wide siRNA screen for regulators of tumor suppressor p53 activity in human non-small cell lung cancer cells identifies components of the RNA splicing machinery as targets for anticancer treatment.

    Science.gov (United States)

    Siebring-van Olst, Ellen; Blijlevens, Maxime; de Menezes, Renee X; van der Meulen-Muileman, Ida H; Smit, Egbert F; van Beusechem, Victor W

    2017-05-01

    Reinstating wild-type tumor suppressor p53 activity could be a valuable option for the treatment of cancer. To contribute to development of new treatment options for non-small cell lung cancer (NSCLC), we performed genome-wide siRNA screens for determinants of p53 activity in NSCLC cells. We identified many genes not previously known to be involved in regulating p53 activity. Silencing p53 pathway inhibitor genes was associated with loss of cell viability. The largest functional gene cluster influencing p53 activity was mRNA splicing. Prominent p53 activation was observed upon silencing of specific spliceosome components, rather than by general inhibition of the spliceosome. Ten genes were validated as inhibitors of p53 activity in multiple NSCLC cell lines: genes encoding the Ras pathway activator SOS1, the zinc finger protein TSHZ3, the mitochondrial membrane protein COX16, and the spliceosome components SNRPD3, SF3A3, SF3B1, SF3B6, XAB2, CWC22, and HNRNPL. Silencing these genes generally increased p53 levels, with distinct effects on CDKN1A expression, induction of cell cycle arrest and cell death. Silencing spliceosome components was associated with alternative splicing of MDM4 mRNA, which could contribute to activation of p53. In addition, silencing splice factors was particularly effective in killing NSCLC cells, albeit in a p53-independent manner. Interestingly, silencing SNRPD3 and SF3A3 exerted much stronger cytotoxicity to NSCLC cells than to lung fibroblasts, suggesting that these genes could represent useful therapeutic targets. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  2. Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-β Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation.

    Directory of Open Access Journals (Sweden)

    Jordan M Meyers

    2017-01-01

    Full Text Available Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.

  3. Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

    Directory of Open Access Journals (Sweden)

    D'Orazi Gabriella

    2008-07-01

    Full Text Available Abstract Background Homeodomain-interacting protein kinase-2 (HIPK2 plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF levels (a β-catenin target gene and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches. Methods The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting. Results HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β activities. Conclusion These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.

  4. Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation.

    Directory of Open Access Journals (Sweden)

    Benjamin M Laitman

    Full Text Available Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1 display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin Kpna4 is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. Kpna4 inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.

  5. Mining protein kinases regulation using graphical models.

    Science.gov (United States)

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2011-03-01

    Abnormal kinase activity is a frequent cause of diseases, which makes kinases a promising pharmacological target. Thus, it is critical to identify the characteristics of protein kinases regulation by studying the activation and inhibition of kinase subunits in response to varied stimuli. Bayesian network (BN) is a formalism for probabilistic reasoning that has been widely used for learning dependency models. However, for high-dimensional discrete random vectors the set of plausible models becomes large and a full comparison of all the posterior probabilities related to the competing models becomes infeasible. A solution to this problem is based on the Markov Chain Monte Carlo (MCMC) method. This paper proposes a BN-based framework to discover the dependency correlations of kinase regulation. Our approach is to apply the MCMC method to generate a sequence of samples from a probability distribution, by which to approximate the distribution. The frequent connections (edges) are identified from the obtained sampling graphical models. Our results point to a number of novel candidate regulation patterns that are interesting in biology and include inferred associations that were unknown.

  6. DMPD: Suppressor of cytokine signaling (SOCS) 2, a protein with multiple functions. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available ns. Rico-Bautista E, Flores-Morales A, Fernandez-Perez L. Cytokine Growth Factor Rev. 2006 Dec;17(6):431-9. ...SOCS) 2, a protein with multiple functions. Authors Rico-Bautista E, Flores-Morales A, Fernandez-Perez L. Pu

  7. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

    DEFF Research Database (Denmark)

    Danovi, Davide; Meulmeester, Erik; Pasini, Diego

    2004-01-01

    Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14(ARF) or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo...

  8. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    Science.gov (United States)

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Expression of ankyrin repeat and suppressor of cytokine signaling box protein 4 (Asb-4) in proopiomelanocortin neurons of the arcuate nucleus of mice produces a hyperphagic, lean phenotype.

    Science.gov (United States)

    Li, Ji-Yao; Chai, Biao-Xin; Zhang, Weizhen; Wang, Hui; Mulholland, Michael W

    2010-01-01

    Ankyrin repeat and suppressor of cytokine signaling box-containing protein 4 (Asb-4) is specifically expressed in the energy homeostasis-related brain areas and colocalizes with proopiomelanocortin (POMC) neurons of the arcuate nucleus (ARC). Injection of insulin into the third ventricle of the rat brain increased Asb-4 mRNA expression in the paraventricular nucleus but not in the ARC of the hypothalamus, whereas injection of leptin (ip) increased Asb-4 expression in both mouse paraventricular nucleus and ARC. A transgenic mouse in which Myc-tagged Asb-4 is specifically expressed in POMC neurons of the ARC was made and used to study the effects of Asb-4 on ingestive behavior and metabolic rate. Animals with overexpression of Asb-4 in POMC neurons demonstrated an increase in food intake. However, POMC-Asb-4 transgenic animals gained significantly less weight from 6-30 wk of age. The POMC-Asb-4 mice had reduced fat mass and increased lean mass and lower levels of blood leptin. The transgenic animals were resistant to high-fat diet-induced obesity. Transgenic mice had significantly higher rates of oxygen consumption and carbon dioxide production than wild-type mice during both light and dark periods. The locomotive activity of transgenic mice was increased. The overexpression of Asb-4 in POMC neurons increased POMC mRNA expression in the ARC. The transgenic animals had no observed effect on peripheral glucose metabolism and the activity of the autonomic nervous system. These results indicate that Asb-4 is a key regulatory protein in the central nervous system, involved in the control of feeding behavior and metabolic rate.

  10. The insulator protein CTCF regulates Drosophila steroidogenesis.

    Science.gov (United States)

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B; Espinàs, M Lluisa

    2015-05-15

    The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. © 2015. Published by The Company of Biologists Ltd.

  11. The insulator protein CTCF regulates Drosophila steroidogenesis

    Directory of Open Access Journals (Sweden)

    Ujué Fresán

    2015-07-01

    Full Text Available The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis.

  12. The molecular effect of metastasis suppressors on Src signaling and tumorigenesis: new therapeutic targets

    Science.gov (United States)

    Liu, Wensheng; Kovacevic, Zaklina; Peng, Zhihai; Jin, Runsen; Wang, Puxiongzhi; Yue, Fei; Zheng, Minhua; Huang, Michael L-H.; Jansson, Patric J.; Richardson, Vera; Kalinowski, Danuta S.; Lane, Darius J.R.; Merlot, Angelica M.; Sahni, Sumit; Richardson, Des R.

    2015-01-01

    A major problem for cancer patients is the metastasis of cancer cells from the primary tumor. This involves: (1) migration through the basement membrane; (2) dissemination via the circulatory system; and (3) invasion into a secondary site. Metastasis suppressors, by definition, inhibit metastasis at any step of the metastatic cascade. Notably, Src is a non-receptor, cytoplasmic, tyrosine kinase, which becomes aberrantly activated in many cancer-types following stimulation of plasma membrane receptors (e.g., receptor tyrosine kinases and integrins). There is evidence of a prominent role of Src in tumor progression-related events such as the epithelial–mesenchymal transition (EMT) and the development of metastasis. However, the precise molecular interactions of Src with metastasis suppressors remain unclear. Herein, we review known metastasis suppressors and summarize recent advances in understanding the mechanisms of how these proteins inhibit metastasis through modulation of Src. Particular emphasis is bestowed on the potent metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1) and its interactions with the Src signaling cascade. Recent studies demonstrated a novel mechanism through which NDRG1 plays a significant role in regulating cancer cell migration by inhibiting Src activity. Moreover, we discuss the rationale for targeting metastasis suppressor genes as a sound therapeutic modality, and we review several examples from the literature where such strategies show promise. Collectively, this review summarizes the essential interactions of metastasis suppressors with Src and their effects on progression of cancer metastasis. Moreover, interesting unresolved issues regarding these proteins as well as their potential as therapeutic targets are also discussed. PMID:26431493

  13. Molecular cloning, phylogenetic analysis, and expression patterns of LATERAL SUPPRESSOR-LIKE and REGULATOR OF AXILLARY MERISTEM FORMATION-LIKE genes in sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Fambrini, Marco; Salvini, Mariangela; Pugliesi, Claudio

    2017-03-01

    The wild sunflower (Helianthus annuus) plants develop a highly branched form with numerous small flowering heads. The origin of a no branched sunflower, producing a single large head, has been a key event in the domestication process of this species. The interaction between hormonal factors and several genes organizes the initiation and outgrowth of axillary meristems (AMs). From sunflower, we have isolated two genes putatively involved in this process, LATERAL SUPPRESSOR (LS)-LIKE (Ha-LSL) and REGULATOR OF AXILLARY MERISTEM FORMATION (ROX)-LIKE (Ha-ROXL), encoding for a GRAS and a bHLH transcription factor (TF), respectively. Typical amino acid residues and phylogenetic analyses suggest that Ha-LSL and Ha-ROXL are the orthologs of the branching regulator LS and ROX/LAX1, involved in the growth habit of both dicot and monocot species. qRT-PCR analyses revealed a high accumulation of Ha-LSL transcripts in roots, vegetative shoots, and inflorescence shoots. By contrast, in internodal stems and young leaves, a lower amount of Ha-LSL transcripts was observed. A comparison of transcription patterns between Ha-LSL and Ha-ROXL revealed some analogies but also remarkable differences; in fact, the gene Ha-ROXL displayed a low expression level in all organs analyzed. In situ hybridization (ISH) analysis showed that Ha-ROXL transcription was strongly restricted to a small domain within the boundary zone separating the shoot apical meristem (SAM) and the leaf primordia and in restricted regions of the inflorescence meristem, beforehand the separation of floral bracts from disc flower primordia. These results suggested that Ha-ROXL may be involved to establish a cell niche for the initiation of AMs as well as flower primordia. The accumulation of Ha-LSL transcripts was not restricted to the boundary zones in vegetative and inflorescence shoots, but the mRNA activity was expanded in other cellular domains of primary shoot apical meristem as well as AMs. In addition, Ha

  14. IRF-8 regulates expansion of myeloid-derived suppressor cells and Foxp3+ regulatory T cells and modulates Th2 immune responses to gastrointestinal nematode infection.

    Directory of Open Access Journals (Sweden)

    Rajesh M Valanparambil

    2017-10-01

    Full Text Available Interferon regulatory factor-8 (IRF-8 is critical for Th1 cell differentiation and negatively regulates myeloid cell development including myeloid-derived suppressor cells (MDSC. MDSC expand during infection with various pathogens including the gastrointestinal (GI nematode Heligmosomoides polygyrus bakeri (Hpb. We investigated if IRF-8 contributes to Th2 immunity to Hpb infection. Irf8 expression was down-regulated in MDSC from Hpb-infected C57BL/6 (B6 mice. IRF-8 deficient Irf8-/- and BXH-2 mice had significantly higher adult worm burdens than B6 mice after primary or challenge Hpb infection. During primary infection, MDSC expanded to a significantly greater extent in mesenteric lymph nodes (MLN and spleens of Irf8-/- and BXH-2 than B6 mice. CD4+GATA3+ T cells numbers were comparable in MLN of infected B6 and IRF-8 deficient mice, but MLN cells from infected IRF-8 deficient mice secreted significantly less parasite-specific IL-4 ex vivo. The numbers of alternatively activated macrophages in MLN and serum levels of Hpb-specific IgG1 and IgE were also significantly less in infected Irf8-/- than B6 mice. The frequencies of antigen-experienced CD4+CD11ahiCD49dhi cells that were CD44hiCD62L- were similar in MLN of infected Irf8-/- and B6 mice, but the proportions of CD4+GATA3+ and CD4+IL-4+ T cells were lower in infected Irf8-/- mice. CD11b+Gr1+ cells from naïve or infected Irf8-/- mice suppressed CD4+ T cell proliferation and parasite-specific IL-4 secretion in vitro albeit less efficiently than B6 mice. Surprisingly, there were significantly more CD4+ T cells in infected Irf8-/- mice, with a higher frequency of CD4+CD25+Foxp3+ T (Tregs cells and significantly higher numbers of Tregs than B6 mice. In vivo depletion of MDSC and/or Tregs in Irf8-/- mice did not affect adult worm burdens, but Treg depletion resulted in higher egg production and enhanced parasite-specific IL-5, IL-13, and IL-6 secretion ex vivo. Our data thus provide a previously

  15. SERPINB5 and AKAP12 -- Expression and promoter methylation of metastasis suppressor genes in pancreatic ductal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Haier Joerg

    2010-10-01

    Full Text Available Abstract Background Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC. Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. Methods Metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. Results In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P SERPINB5 mRNA expression was correlated with increased metastasis scores (P SERPINB5 methylation was associated with loss of mRNA and protein expression (P SERPINB5 methylation was also directly correlated to decreased metastasis scores (P Conclusions AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

  16. Autopalmitoylation of TEAD proteins regulates transcriptional output of the Hippo pathway.

    Science.gov (United States)

    Chan, PuiYee; Han, Xiao; Zheng, Baohui; DeRan, Michael; Yu, Jianzhong; Jarugumilli, Gopala K; Deng, Hua; Pan, Duojia; Luo, Xuelian; Wu, Xu

    2016-04-01

    TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

  17. DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERα-positive breast cancer cells

    NARCIS (Netherlands)

    M. Ansems (Marleen); J.N. Søndergaard (Jonas Nørskov); A.M. Sieuwerts (Anieta); M.W.G. Looman (Maaike W. G.); M. Smid (Marcel); A.M.A. de Graaf (Annemarie M. A.); V. de Weerd (Vanja); M. Zuidscherwoude (Malou); J.A. Foekens (John); J.W.M. Martens (John); G.J. Adema (Gosse J.)

    2015-01-01

    textabstractBreast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERα) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERα and as a strong and independent

  18. DC-SCRIPT is a novel regulator of the tumor suppressor gene CDKN2B and induces cell cycle arrest in ERalpha-positive breast cancer cells

    NARCIS (Netherlands)

    Ansems, M.; Sondergaard, J.N.; Sieuwerts, A.M.; Looman, M.W.G.; Smid, M.; Graaf, A.M.A. de; Weerd, V. de; Zuidscherwoude, M.; Foekens, J.A.; Martens, J.W.; Adema, G.J.

    2015-01-01

    Breast cancer is one of the most common causes of cancer-related deaths in women. The estrogen receptor (ERalpha) is well known for having growth promoting effects in breast cancer. Recently, we have identified DC-SCRIPT (ZNF366) as a co-suppressor of ERalpha and as a strong and independent

  19. Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

    DEFF Research Database (Denmark)

    Kazi, Julhash U; Sun, Jianmin; Phung, Bengt

    2012-01-01

    The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases...

  20. Expression of von Hippel-Lindau tumor suppressor protein (pVHL) characteristic of tongue cancer and proliferative lesions in tongue epithelium.

    Science.gov (United States)

    Hasegawa, Hisashi; Kusumi, Yoshiaki; Asakawa, Takeshi; Maeda, Miyoko; Oinuma, Toshinori; Furusaka, Tohru; Oshima, Takeshi; Esumi, Mariko

    2017-05-26

    Patients with tongue cancer frequently show loss of heterozygosity (LOH) of the von Hippel-Lindau (VHL) tumor suppressor gene. However, expression of VHL protein (pVHL) in tongue cancer has rarely been investigated and remains largely unknown. We performed immunohistochemical staining of pVHL in tongue tissues and dysplasia, and examined the association with LOH and its clinical significance. Immunohistochemical staining of pVHL in formalin-fixed, paraffin-embedded sections of cancerous and other tissues from 19 tongue cancer patients showed positivity for LOH of VHL in four samples, negativity in four samples, and was non-informative in 11 samples. The staining pattern of pVHL was also compared with those of cytokeratin (CK) 13 and CK17. In normal tongue tissues, pVHL staining was localized to the cytoplasm of cells in the basal layer and the area of the spinous layer adjacent to the basal layer of stratified squamous epithelium. Positive staining for pVHL was observed in the cytoplasm of cancer cells from all 19 tongue cancer patients. No differences as a result of the presence or absence of LOH were found. Notably, cytoplasm of poorly differentiated invasive cancer cells was less intensely stained than that of well and moderately differentiated invasive cancer cells. pVHL staining was also evident in epithelial dysplasia lesions with pVHL-positive cells expanding from the basal layer to the middle of the spinous layer. However, no CK13 staining was noted in regions of the epithelium, which were positive for pVHL. In contrast, regions with positive staining for CK17 closely coincided with those positive for pVHL. Positive staining for pVHL was observed in cancerous areas but not in normal tissues. pVHL expression was also detected in lesions of epithelial dysplasia. These findings suggest that pVHL may be a useful marker for proliferative lesions.

  1. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  2. Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation.

    Science.gov (United States)

    Koshibu, K; Gräff, J; Mansuy, I M

    2011-01-26

    Complex brain diseases and neurological disorders in human generally result from the disturbance of multiple genes and signaling pathways. These disturbances may derive from mutations, deletions, translocations or rearrangements of specific gene(s). However, over the past years, it has become clear that such disturbances may also derive from alterations in the epigenome affecting several genes simultaneously. Our work recently demonstrated that epigenetic mechanisms in the adult brain are in part regulated by protein phosphatase 1 (PP1), a protein Ser/Thr phosphatase that negatively regulates hippocampus-dependent long-term memory (LTM) and synaptic plasticity. PP1 is abundant in brain structures involved in emotional processing like the amygdala, it may therefore be involved in the regulation of fear memory, a form of memory related to post-traumatic stress disorder (PTSD) in human. Here, we demonstrate that PP1 is a molecular suppressor of fear memory and synaptic plasticity in the amygdala that can control chromatin remodeling in neurons. We show that the selective inhibition of the nuclear pool of PP1 in amygdala neurons significantly alters posttranslational modifications (PTMs) of histones and the expression of several memory-associated genes. These alterations correlate with enhanced fear memory, and with an increase in long-term potentiation (LTP) that is transcription-dependent. Our results underscore the importance of nuclear PP1 in the amygdala as an epigenetic regulator of emotional memory, and the relevance of protein phosphatases as potential targets for therapeutic treatment of brain disorders like PTSD. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. S100 Proteins As an Important Regulator of Macrophage Inflammation

    Directory of Open Access Journals (Sweden)

    Chang Xia

    2018-01-01

    Full Text Available The S100 proteins, a family of calcium-binding cytosolic proteins, have a broad range of intracellular and extracellular functions through regulating calcium balance, cell apoptosis, migration, proliferation, differentiation, energy metabolism, and inflammation. The intracellular functions of S100 proteins involve interaction with intracellular receptors, membrane protein recruitment/transportation, transcriptional regulation and integrating with enzymes or nucleic acids, and DNA repair. The S100 proteins could also be released from the cytoplasm, induced by tissue/cell damage and cellular stress. The extracellular S100 proteins, serving as a danger signal, are crucial in regulating immune homeostasis, post-traumatic injury, and inflammation. Extracellular S100 proteins are also considered biomarkers for some specific diseases. In this review, we will discuss the multi-functional roles of S100 proteins, especially their potential roles associated with cell migration, differentiation, tissue repair, and inflammation.

  4. KF-1 ubiquitin ligase: anxiety suppressor model.

    Science.gov (United States)

    Hashimoto-Gotoh, Tamotsu; Iwabe, Naoyuki; Tsujimura, Atsushi; Nakagawa, Masanori; Marunaka, Yoshinori

    2011-06-01

    Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.

  5. Post-transcriptional modulation of protein phosphatase PPP2CA and tumor suppressor PTEN by endogenous siRNA cleaved from hairpin within PTEN mRNA 3'UTR in human liver cells.

    Science.gov (United States)

    Gao, Yu-En; Wang, Yuan; Chen, Fu-Quan; Feng, Jin-Yan; Yang, Guang; Feng, Guo-Xing; Yang, Zhe; Ye, Li-Hong; Zhang, Xiao-Dong

    2016-07-01

    Increasing evidence shows that mRNAs exert regulatory function along with coding proteins. Recently we report that a hairpin within YAP mRNA 3'UTR can modulate the Hippo signaling pathway. PTEN is a tumor suppressor, and is mutated in human cancers. In this study we examined whether PTEN mRNA 3'UTR contained a hairpin structure that could regulate gene regulation at the post-transcriptional level. The secondary structure of PTEN mRNA 3'UTR was analyzed using RNAdraw and RNAstructure. Function of hairpin structure derived from the PTEN mRNA 3'UTR was examined using luciferase reporter assay, RT-PCR and Western blotting. RNA-immunoprecipitation (RIP) assay was used to analyze the interaction between PTEN mRNA and microprocessor Drosha and DGCR8. Endogenous siRNA (esiRNA) derived from PTEN mRNA 3'UTR was identified by RT-PCR and rt-PCR, and its target genes were predicted using RNAhybrid. A bioinformatics analysis revealed that PTEN mRNA contained a hairpin structure (termed PTEN-sh) within 3'UTR, which markedly increased the reporter activities of AP-1 and NF-κB in 293T cells. Moreover, treatment with PTEN-sh (1 and 2 μg) dose-dependently inhibited the expression of PTEN in human liver L-O2 cells. RIP assay demonstrated that the microprocessor Drosha and DGCR8 was bound to PTEN-sh in L-O2 cells, leading to the cleavage of PTEN-sh from PTEN mRNA 3'UTR. In addition, microprocessor Dicer was involved in the processing of PTEN-sh. Interestingly, esiRNA (termed PTEN-sh-3p21) cleaved from PTEN-sh was identified in 293T cells and human liver tissues, which was found to target the mRNA 3'UTRs of protein phosphatase PPP2CA and PTEN in L-O2 cells. Treatment of L-O2 or Chang liver cells with PTEN-sh-3p21 (50, 100 nmol/L) promoted the cell proliferation in dose- and time-dependent manners. The endogenous siRNA (PTEN-sh-3p21) cleaved from PTEN-sh within PTEN mRNA 3'UTR modulates PPP2CA and PTEN at the post-transcriptional level in liver cells.

  6. Post-transcriptional modulation of protein phosphatase PPP2CA and tumor suppressor PTEN by endogenous siRNA cleaved from hairpin within PTEN mRNA 3′UTR in human liver cells

    Science.gov (United States)

    Gao, Yu-en; Wang, Yuan; Chen, Fu-quan; Feng, Jin-yan; Yang, Guang; Feng, Guo-xing; Yang, Zhe; Ye, Li-hong; Zhang, Xiao-dong

    2016-01-01

    Aim: Increasing evidence shows that mRNAs exert regulatory function along with coding proteins. Recently we report that a hairpin within YAP mRNA 3′UTR can modulate the Hippo signaling pathway. PTEN is a tumor suppressor, and is mutated in human cancers. In this study we examined whether PTEN mRNA 3′UTR contained a hairpin structure that could regulate gene regulation at the post-transcriptional level. Methods: The secondary structure of PTEN mRNA 3′UTR was analyzed using RNAdraw and RNAstructure. Function of hairpin structure derived from the PTEN mRNA 3′UTR was examined using luciferase reporter assay, RT-PCR and Western blotting. RNA-immunoprecipitation (RIP) assay was used to analyze the interaction between PTEN mRNA and microprocessor Drosha and DGCR8. Endogenous siRNA (esiRNA) derived from PTEN mRNA 3′UTR was identified by RT-PCR and rt-PCR, and its target genes were predicted using RNAhybrid. Results: A bioinformatics analysis revealed that PTEN mRNA contained a hairpin structure (termed PTEN-sh) within 3′UTR, which markedly increased the reporter activities of AP-1 and NF-κB in 293T cells. Moreover, treatment with PTEN-sh (1 and 2 μg) dose-dependently inhibited the expression of PTEN in human liver L-O2 cells. RIP assay demonstrated that the microprocessor Drosha and DGCR8 was bound to PTEN-sh in L-O2 cells, leading to the cleavage of PTEN-sh from PTEN mRNA 3′UTR. In addition, microprocessor Dicer was involved in the processing of PTEN-sh. Interestingly, esiRNA (termed PTEN-sh-3p21) cleaved from PTEN-sh was identified in 293T cells and human liver tissues, which was found to target the mRNA 3′UTRs of protein phosphatase PPP2CA and PTEN in L-O2 cells. Treatment of L-O2 or Chang liver cells with PTEN-sh-3p21 (50, 100 nmol/L) promoted the cell proliferation in dose- and time-dependent manners. Conclusion: The endogenous siRNA (PTEN-sh-3p21) cleaved from PTEN-sh within PTEN mRNA 3′UTR modulates PPP2CA and PTEN at the post

  7. Regulation of the PML tumor suppressor in drug-induced senescence of human normal and cancer cells by JAK/STAT-mediated signaling

    Czech Academy of Sciences Publication Activity Database

    Hubáčková, Soňa; Nováková, Zora; Krejčíková, Kateřina; Košař, Martin; Dobrovolná, Jana; Dušková, Pavlína; Hanzlíková, Hana; Vančurová, Markéta; Barath, P.; Bartek, J.; Hodný, Zdeněk

    2010-01-01

    Roč. 9, č. 15 (2010), s. 3085-3099 ISSN 1538-4101 R&D Projects: GA AV ČR IAA500390501; GA ČR GA204/08/1418; GA ČR GA301/08/0353 Institutional research plan: CEZ:AV0Z50520514 Keywords : PML tumor suppressor * cellular senescence * JAK-STAT signaling pathway Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.999, year: 2010

  8. Tumor suppressor identified as inhibitor of inflammation

    Science.gov (United States)

    Scientists at NCI have found that a protein, FBXW7, which acts as a tumor suppressor, is also important for the reduction in strength of inflammatory pathways. It has long been recognized that a complex interaction exists between cancer causing mechanisms

  9. RNAi suppressors encoded by pathogenic human viruses

    NARCIS (Netherlands)

    de Vries, Walter; Berkhout, Ben

    2008-01-01

    RNA silencing or RNAi interference (RNAi) serves as an innate antiviral mechanism in plants, fungi and animals. Human viruses, like plant viruses, encode suppressor proteins or RNAs that block or modulate the RNAi pathway. This review summarizes the mechanisms by which pathogenic human viruses

  10. Modulator of Apoptosis 1: A Highly Regulated RASSF1A-Interacting BH3-Like Protein.

    Science.gov (United States)

    Law, Jennifer; Yu, Victor C; Baksh, Shairaz

    2012-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in both the intrinsic and extrinsic modes of cell death or apoptosis. MOAP-1 is part of the Ras association domain family 1A (RASSF1A)/MOAP-1 pro-apoptotic extrinsic signaling pathway that regulates apoptosis by utilizing death receptors such as tumor necrosis factor α (TNFα) or TNF-related apoptosis-inducing ligand (TRAIL) to inhibit abnormal growth. RASSF1A is a bona fide tumor suppressor gene that is epigenetically silenced by promoter-specific methylation in numerous human cancers. MOAP-1 is a downstream effector of RASSF1A that promotes Bax activation and cell death and is highly regulated during apoptosis. We speculate that MOAP-1 and RASSF1A are important elements of an "apoptotic checkpoint" that directly influences the outcome of cell death. The failure to regulate this pro-apoptotic pathway may result in the appearance of cancer and possibly other disorders. Although loss of RASSF1A expression is frequently observed in human cancers, it is currently unknown if MOAP-1 expression may also be affected during carcinogenesis to result in uncontrolled malignant growth. In this article, we will summarize what is known about the biological role(s) of MOAP-1 and how it functions as a downstream effector to RASSF1A.

  11. Regulation, Signaling, and Physiological Functions of G-Proteins.

    Science.gov (United States)

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. RASSF6; the Putative Tumor Suppressor of the RASSF Family

    Directory of Open Access Journals (Sweden)

    Hiroaki Iwasa

    2015-12-01

    Full Text Available Humans have 10 genes that belong to the Ras association (RA domain family (RASSF. Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1 are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.

  13. RASSF6; the Putative Tumor Suppressor of the RASSF Family.

    Science.gov (United States)

    Iwasa, Hiroaki; Jiang, Xinliang; Hata, Yutaka

    2015-12-09

    Humans have 10 genes that belong to the Ras association (RA) domain family (RASSF). Among them, RASSF7 to RASSF10 have the RA domain in the N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ)-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo) and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1) are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.

  14. ABCE1 is a highly conserved RNA silencing suppressor.

    Directory of Open Access Journals (Sweden)

    Kairi Kärblane

    Full Text Available ATP-binding cassette sub-family E member 1 (ABCE1 is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference.

  15. Regulation of intestinal protein metabolism by amino acids.

    Science.gov (United States)

    Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

    2013-09-01

    Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

  16. Transducer of ERBB2.1 (TOB1 as a Tumor Suppressor: A Mechanistic Perspective

    Directory of Open Access Journals (Sweden)

    Hun Seok Lee

    2015-12-01

    Full Text Available Transducer of ERBB2.1 (TOB1 is a tumor-suppressor protein, which functions as a negative regulator of the receptor tyrosine-kinase ERBB2. As most of the other tumor suppressor proteins, TOB1 is inactivated in many human cancers. Homozygous deletion of TOB1 in mice is reported to be responsible for cancer development in the lung, liver, and lymph node, whereas the ectopic overexpression of TOB1 shows anti-proliferation, and a decrease in the migration and invasion abilities on cancer cells. Biochemical studies revealed that the anti-proliferative activity of TOB1 involves mRNA deadenylation and is associated with the reduction of both cyclin D1 and cyclin-dependent kinase (CDK expressions and the induction of CDK inhibitors. Moreover, TOB1 interacts with an oncogenic signaling mediator, β-catenin, and inhibits β-catenin-regulated gene transcription. TOB1 antagonizes the v-akt murine thymoma viral oncogene (AKT signaling and induces cancer cell apoptosis by activating BCL2-associated X (BAX protein and inhibiting the BCL-2 and BCL-XL expressions. The tumor-specific overexpression of TOB1 results in the activation of other tumor suppressor proteins, such as mothers against decapentaplegic homolog 4 (SMAD4 and phosphatase and tensin homolog-10 (PTEN, and blocks tumor progression. TOB1-overexpressing cancer cells have limited potential of growing as xenograft tumors in nude mice upon subcutaneous implantation. This review addresses the molecular basis of TOB1 tumor suppressor function with special emphasis on its regulation of intracellular signaling pathways.

  17. End-Binding Protein 1 (EB1) Up-regulation is an Early Event in Colorectal Carcinogenesis

    Science.gov (United States)

    Stypula-Cyrus, Yolanda; Mutyal, Nikhil N.; Cruz, Mart Angelo Dela; Kunte, Dhananjay P.; Radosevich, Andrew J.; Wali, Ramesh; Roy, Hemant K.; Backman, Vadim

    2014-01-01

    End-binding protein (EB1) is a microtubule protein that binds to the tumor suppressor adenomatous polyposis coli (APC). While EB1 is implicated as a potential oncogene, its role in cancer progression is unknown. Therefore, we analyzed EB1/APC expression at the earliest stages of colorectal carcinogenesis and in the uninvolved mucosa ("field effect") of human and animal tissue. We also performed siRNA-knockdown in colon cancer cell lines. EB1 is up-regulated in early and field carcinogenesis in the colon, and the cellular/nano-architectural effect of EB1 knockdown depended on the genetic context. Thus, dysregulation of EB1 is an important early event in colon carcinogenesis. PMID:24492008

  18. Phosphorylation-regulated degradation of the tumor-suppressor form of PED by chaperone-mediated autophagy in lung cancer cells.

    Science.gov (United States)

    Quintavalle, Cristina; Di Costanzo, Stefania; Zanca, Ciro; Tasset, Immaculada; Fraldi, Alessandro; Incoronato, Mariarosaria; Mirabelli, Peppino; Monti, Maria; Ballabio, Andrea; Pucci, Piero; Cuervo, Ana Maria; Condorelli, Gerolama

    2014-10-01

    PED/PEA-15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)-which, among other processes, is involved in chaperone-mediated autophagy (CMA)-as a PED-interacting protein. We found that PED has two CMA-like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ-like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up-regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms. © 2014 Wiley Periodicals, Inc.

  19. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication

    DEFF Research Database (Denmark)

    Piunti, Andrea; Rossi, Alessandra; Cerutti, Aurora

    2014-01-01

    The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human...

  20. Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity.

    Science.gov (United States)

    Gerber, Kyle J; Squires, Katherine E; Hepler, John R

    2016-02-01

    The regulator of G protein signaling (RGS) family of proteins serves critical roles in G protein-coupled receptor (GPCR) and heterotrimeric G protein signal transduction. RGS proteins are best understood as negative regulators of GPCR/G protein signaling. They achieve this by acting as GTPase activating proteins (GAPs) for Gα subunits and accelerating the turnoff of G protein signaling. Many RGS proteins also bind additional signaling partners that either regulate their functions or enable them to regulate other important signaling events. At neuronal synapses, GPCRs, G proteins, and RGS proteins work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity, which are necessary for central nervous system physiology and behavior. Accumulating evidence has revealed key roles for specific RGS proteins in multiple signaling pathways at neuronal synapses, regulating both pre- and postsynaptic signaling events and synaptic plasticity. Here, we review and highlight the current knowledge of specific RGS proteins (RGS2, RGS4, RGS7, RGS9-2, and RGS14) that have been clearly demonstrated to serve critical roles in modulating synaptic signaling and plasticity throughout the brain, and we consider their potential as future therapeutic targets. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. Suppressor of cytokine signaling 2 (SOCS2) deletion protects against multiple low dose streptozotocin-induced type 1 diabetes in adult male mice

    DEFF Research Database (Denmark)

    Alkharusi, Amira; Mirecki-Garrido, Mercedes; Ma, Zuheng

    2016-01-01

    Background: Diabetes type 1 is characterized by the failure of beta cells to produce insulin. Suppressor of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Previous studies have shown that GH can ...

  2. ASIC PROTEINS REGULATE SMOOTH MUSCLE CELL MIGRATION

    OpenAIRE

    Grifoni, Samira C.; Jernigan, Nikki L.; Hamilton, Gina; Drummond, Heather A.

    2007-01-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated Epithelial Na+ Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration, however the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence indi...

  3. Evaluating Modulators of ‘Regulator of G-protein Signaling’ (RGS) Proteins

    OpenAIRE

    Bosch, Dustin E.; Zielinski, Thomas; Lowery, Robert G.; Siderovski, David P.

    2012-01-01

    Regulator of G-protein Signaling’ (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for small molecule modulation. Described in this unit are high-throughput screening procedures for identifying modulators of RGS protein-mediated GTPase acceleration (‘GAP activity’), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is...

  4. Copper delivery to chloroplast proteins and its regulation

    Directory of Open Access Journals (Sweden)

    Guadalupe eAguirre

    2016-01-01

    Full Text Available Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase,¬ but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.

  5. ASIC proteins regulate smooth muscle cell migration.

    Science.gov (United States)

    Grifoni, Samira C; Jernigan, Nikki L; Hamilton, Gina; Drummond, Heather A

    2008-03-01

    The purpose of the present study was to investigate Acid Sensing Ion Channel (ASIC) protein expression and importance in cellular migration. We recently demonstrated that Epithelial Na(+)Channel (ENaC) proteins are required for vascular smooth muscle cell (VSMC) migration; however, the role of the closely related ASIC proteins has not been addressed. We used RT-PCR and immunolabeling to determine expression of ASIC1, ASIC2, ASIC3 and ASIC4 in A10 cells. We used small interference RNA to silence individual ASIC expression and determine the importance of ASIC proteins in wound healing and chemotaxis (PDGF-bb)-initiated migration. We found ASIC1, ASIC2, and ASIC3, but not ASIC4, expression in A10 cells. ASIC1, ASIC2, and ASIC3 siRNA molecules significantly suppressed expression of their respective proteins compared to non-targeting siRNA (RISC) transfected controls by 63%, 44%, and 55%, respectively. Wound healing was inhibited by 10, 20, and 26% compared to RISC controls following suppression of ASIC1, ASIC2, and ASIC3, respectively. Chemotactic migration was inhibited by 30% and 45%, respectively, following suppression of ASIC1 and ASIC3. ASIC2 suppression produced a small, but significant, increase in chemotactic migration (4%). Our data indicate that ASIC expression is required for normal migration and may suggest a novel role for ASIC proteins in cellular migration.

  6. The Drosophila secreted protein Argos regulates signal transduction in the Ras/MAPK pathway.

    Science.gov (United States)

    Sawamoto, K; Okabe, M; Tanimura, T; Mikoshiba, K; Nishida, Y; Okano, H

    1996-08-25

    The Drosophila argos gene encodes a secreted protein with an EGF motif which acts as an inhibitor of cellular differentiation in multiple developmental processes. To investigate the cellular pathways regulated by Argos, we screened for mutations which could modify the phenotype caused by overexpression of argos. We show that the effects of argos overexpression on the eye and wing vein development are suppressed by gain-of-function mutations of the MAPKK/D-MEK gene (Dsor1/D-mek) and the MAPK/ERK-A gene (rolled) and were enhanced by loss-of-function mutations of Star. Loss-of-function mutations in components of the Ras/MAPK signaling cascade act as dominant suppressors of the phenotype caused by the argos null mutations. A loss-of-function argos mutation enhanced the overproduction of R7 neurons caused by gain-of-function alleles of Son of sevenless and Dsor1. Conversely, overexpression of argos inhibited formation of the extra R7 cells that was caused by high-level MAPK/ERK-A activity. A phenotype of the sev; argos double mutants revealed that sev is epistatic to argos. These results provide evidence that Argos negatively regulates signal transduction events in the Ras/MAPK cascade.

  7. SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation.

    Science.gov (United States)

    Park, Seung Kuk; Jeong, Sunjoo

    2016-02-05

    Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2016-02-05

    Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

  9. The Human IL-22 Receptor Is Regulated through the Action of the Novel E3 Ligase Subunit FBXW12, Which Functions as an Epithelial Growth Suppressor

    Directory of Open Access Journals (Sweden)

    Joseph Franz

    2015-01-01

    Full Text Available Interleukin- (IL- 22 signaling is protective in animal models of pneumonia and bacteremia by Klebsiella pneumoniae and mediates tissue recovery from influenza and Staph aureus infection. We recently described processing of mouse lung epithelial IL-22 receptor (IL-22R by ubiquitination on the intracellular C-terminal. To identify cellular factors that regulate human IL-22R, we screened receptor abundance while overexpressing constituents of the ubiquitin system and identify that IL-22R can be shuttled for degradation by multiple previously uncharacterized F-box protein E3 ligase subunits. We observe that in human cells IL-22R is destabilized by FBXW12. FBXW12 causes depletion of endogenous and plasmid-derived IL-22R in lung epithelia, binds the E3 ligase constituent Skp-1, and facilitates ubiquitination of IL-22R in vitro. FBXW12 knockdown with shRNA increases IL-22R abundance and STAT3 phosphorylation in response to IL-22 cytokine treatment. FBXW12 shRNA increases human epithelial cell growth and cell cycle progression with enhanced constitutive activity of map kinases JNK and ERK. These findings indicate that the heretofore-undescribed protein FBXW12 functions as an E3 ligase constituent to ubiquitinate and degrade IL-22R and that therapeutic FBXW12 inhibition may enhance IL-22 signaling and bolster mucosal host defense and infection containment.

  10. Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    An, Joo-Hee; Kim, Jung-Woong; Jang, Sang-Min; Kim, Chul-Hong; Kang, Eun-Jin; Choi, Kyung-Hee, E-mail: khchoi@cau.ac.kr

    2011-08-19

    Highlights: {yields} The actin binding protein Gelsolin (GSN) interacts with transcription factor p53. {yields} GSN interacts with transactivation- and DNA binding domains of p53. {yields} GSN represses transactivity of p53 via inhibition of nuclear translocation of p53. {yields} GSN inhibits the p53-mediated apoptosis in hepatocarcinoma HepG2 cells. -- Abstract: As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

  11. Dynamic regulation of uncoupling protein 2 expression by microRNA-214 in hepatocellular carcinoma.

    Science.gov (United States)

    Yu, Guangsheng; Wang, Jianlu; Xu, Kesen; Dong, Jiahong

    2016-07-01

    Gemcitabine (GEM), a commonly used chemotherapeutic agent in hepatocellular carcinoma (HCC) patients, uses oxidative stress induction as a common effector pathway. However, GEM alone or in combination with oxaliplatin hardly renders any survival benefits to HCC patients. We have recently shown that this is part due to the overexpression of the mitochondrial uncoupling protein 2 (UCP2) that in turn mediates resistance to GEM in HCC patients. However, not much is known about regulatory mechanisms underlying UCP2 overexpression in HCC. Differential protein expression in HCC cell lines did not show a concomitant change in UCP2 transcript level, indicating post-transcriptional or post-translational regulatory mechanism. In situ analysis revealed that UCP2 is a putative target of miR-214 miR-214 expression is significantly down-regulated in HCC patient samples as compared with normal adjacent tissues and in cell line, human hepatoblastoma cells (HuH6), with high UCP2 protein expression. We demonstrated using miR-214 mimic and antagomir that the miRNA targeted UCP2 expression by directly targeting the wild-type, but not a miR-214 seed mutant, 3' UTR of UCP2 Overexpression of miR-214 significantly attenuated cell proliferation. Finally, analysis in 20 HCC patients revealed an inverse correlation in expression of UCP2 and miR-214 (Pearson's correlation coefficient, r=-0.9792). Cumulatively, our data indicate that in the context of HCC, miR-214 acts as a putative tumour suppressor by targeting UCP2 and defines a novel mechanism of regulation of UCP2. © 2016 The Author(s).

  12. KeaA, a Dictyostelium kelch-domain protein that regulates the response to stress and development

    Directory of Open Access Journals (Sweden)

    Souza Glaucia M

    2010-07-01

    Full Text Available Abstract Background The protein kinase YakA is responsible for the growth arrest and induction of developmental processes that occur upon starvation of Dictyostelium cells. yakA- cells are aggregation deficient, have a faster cell cycle and are hypersensitive to oxidative and nitrosoative stress. With the aim of isolating members of the YakA pathway, suppressors of the death induced by nitrosoative stress in the yakA- cells were identified. One of the suppressor mutations occurred in keaA, a gene identical to DG1106 and similar to Keap1 from mice and the Kelch protein from Drosophila, among others that contain Kelch domains. Results A mutation in keaA suppresses the hypersensitivity to oxidative and nitrosoative stresses but not the faster growth phenotype of yakA- cells. The growth profile of keaA deficient cells indicates that this gene is necessary for growth. keaA deficient cells are more resistant to nitrosoative and oxidative stress and keaA is necessary for the production and detection of cAMP. A morphological analysis of keaA deficient cells during multicellular development indicated that, although the mutant is not absolutely deficient in aggregation, cells do not efficiently participate in the process. Gene expression analysis using cDNA microarrays of wild-type and keaA deficient cells indicated a role for KeaA in the regulation of the cell cycle and pre-starvation responses. Conclusions KeaA is required for cAMP signaling following stress. Our studies indicate a role for kelch proteins in the signaling that regulates the cell cycle and development in response to changes in the environmental conditions.

  13. Regulation of gap junctions by protein phosphorylation

    Directory of Open Access Journals (Sweden)

    J.C. Sáez

    1998-05-01

    Full Text Available Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

  14. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...

  15. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression

    DEFF Research Database (Denmark)

    Dhamrait, Sukhbir S.; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik

    2016-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial...

  16. Regulation of Cellular and Molecular Functions by Protein ...

    Indian Academy of Sciences (India)

    critical role in the regulation of many cellular functions. Phosphorylation of proteins is carried out by a group of enzymes known as protein kinases which transfer terminal phosphate of. ATP (sometimes GTP) to the hydroxyl group of amino acids - serine, threonine and tyrosine. Phosphate group on these three hydroxy amino ...

  17. Claudins, dietary milk proteins, and intestinal barrier regulation.

    Science.gov (United States)

    Kotler, Belinda M; Kerstetter, Jane E; Insogna, Karl L

    2013-01-01

    The family of claudin proteins plays an important role in regulating the intestinal barrier by modulating the permeability of tight junctions. The impact of dietary protein on claudin biology has not been studied extensively. Whey proteins have been reported to improve intestinal barrier function, but their mechanism of action is not clear. Recent studies, however, have demonstrated increased intestinal claudin expression in response to milk protein components. Reviewed here are new findings suggesting that whey-protein-derived transforming growth factor β transcriptionally upregulates claudin-4 expression via a Smad-4-dependent pathway. These and other data, including limited clinical studies, are summarized below and, in the aggregate, suggest a therapeutic role for whey protein in diseases of intestinal barrier dysfunction, perhaps, in part, by regulating claudin expression. © 2013 International Life Sciences Institute.

  18. Down-regulation of PTEN by HCV core protein through activating nuclear factor-κB.

    Science.gov (United States)

    Zhang, Yong; Li, Rong-Qing; Feng, Xu-Dong; Zhang, Yan-Hua; Wang, Li

    2014-01-01

    The hepatitis C virus (HCV) core protein is an important causative agent in HCV related hepatocellular carcinoma (HCC). Tumor suppressor gene PTEN appears to act in the liver at the crossroad of processes controlling cell proliferation. In this study we investigated the effect of the HCV core protein on the PTEN pathway in hepatocarcinogenesis. The HCV core was transfected stably into HepG2 cell. The effect of HCV core on cell proliferation and viability were detected by 3-(4, 5)-dimethylthiahiazo-(-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay, clonogenic survival assay and Fluorescence Activating Cell Sorter (FACS) analysis. The expressions of PTEN were detected by real time RT-PCR and/or Western blot analysis, also the mechanism of down-regulation of PTEN was explored by western blot, luciferase assay and RNA interference. We found the HCV core promoted cell proliferation, survival and G2/M phase accumulation. It downregulated PTEN at mRNA and protein level and activated PTEN downstream gene Akt accompanied with NF-κB activation. Furthermore, the inhibition of HCV core by its specific shRNAs decreased the effect of growth promotion and G2/M phase arrest, inhibited the expression of nuclear p65 and increased PTEN expression. The activity of PTEN was restored when treated with NF-κB inhibitor PDTC. By luciferase assay we found that NF-κB inhibited PTEN promoter transcription activity directly in HCV core cells, while PDTC was contrary. Our study suggests that HCV proteins could modulate PTEN by activating NF-κB. Furthermore strategies designed to restore the expression of PTEN may be promising therapies for preventing HCV dependent hepatocarcinogenesis.

  19. Allospecific CD8 T suppressor cells induced by multiple MLC stimulation or priming in the presence of ILT3.Fc have similar gene expression profiles.

    Science.gov (United States)

    Chen, Ling; Xu, Zheng; Chang, Chris; Ho, Sophey; Liu, Zhuoru; Vlad, George; Cortesini, Raffaello; Clynes, Raphael A; Luo, Yun; Suciu-Foca, Nicole

    2014-02-01

    Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8(+) CD28(-) T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity. Copyright © 2014. Published by Elsevier Inc.

  20. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  1. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  2. Tumour suppressor PTEN regulates cell cycle and protein kinase B/Akt pathway in breast cancer cells

    Czech Academy of Sciences Publication Activity Database

    Hlobilková, Alice; Knillová, J.; Šváchová, M.; Skypalová, P.; Kryštof, Vladimír; Kolář, Z.

    2006-01-01

    Roč. 26, 2A (2006), s. 1015-1022 ISSN 0250-7005 R&D Projects: GA MZd NR7828 Institutional research plan: CEZ:AV0Z50380511 Keywords : breast cancer cell lines * cell cycle * phosphatase activity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.479, year: 2006

  3. Immune regulation of Rab proteins expression and intracellular transport.

    Science.gov (United States)

    Pei, Gang; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel

    2012-07-01

    Compartmentalization in cells of the immune system, the focus of this review, facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In this process of compartment maintenance, Rab proteins are central regulators of protein-mediated transport and fusion of intracellular structures. It is widely believed that the intracellular concentration of proteins that regulate intracellular transport, including Rab proteins, is constitutively mantained. However, there is a growing body of evidence indicating that transcriptional rates of Rab proteins can be modified. This process is especially evident during immune activation and argues that after activation, these cells require higher levels of Rab proteins. The aim of this review is to discuss evidence showing the increasing links between Rab protein expression and intracellular transport, particularly in monocytes and macrophages. We highlight here biological processes in which the expression of Rab GTPases is selectively regulated, leading to the activation of specific intracellular routes. Further, we focus on the immune regulation of intracellular transport after cytokine activation and microbial infection, with an emphasis in mycobacterial infection.

  4. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    Science.gov (United States)

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. Copyright © 2016. Published by Elsevier Ltd.

  5. Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.

    Science.gov (United States)

    Kilander, Michaela B C; Petersen, Julian; Andressen, Kjetil Wessel; Ganji, Ranjani Sri; Levy, Finn Olav; Schuster, Jens; Dahl, Niklas; Bryja, Vitezslav; Schulte, Gunnar

    2014-05-01

    Frizzleds (FZDs) are classified as G-protein-coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD6 regulates convergent extension movements, and its C-terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD6, Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD6 precouples to Gαi1 and Gαq but not to GαoA,Gαs, and Gα12 proteins. G-protein coupling is measured as a 10-20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross-linking. The FZD6 Arg511Cys mutation is incapable of G-protein precoupling, even though it still binds DVL. Using both FRAP and Förster resonance energy transfer (FRET) technology, we showed that the FZD6-Gαi1 and FZD-Gαq complexes dissociate on WNT-5A stimulation. Most important, G-protein precoupling of FZD6 and WNT-5A-induced signaling to extracellular signal-regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD6-G-protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G-protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G-protein, and DVL function.

  6. The putative tumor suppressor, miR-199a, regulated by Snail, modulates clear cell renal cell carcinoma aggressiveness by repressing ROCK1.

    Science.gov (United States)

    Zhang, Xiao; Li, Peng; Ding, Zhen; Wang, Huili; Wang, Junye; Han, Lei; Ding, Shangwei

    2018-01-01

    Aberrant expression of miR-199a has been frequently reported in cancer studies; however, its role in renal cell carcinoma (RCC) has not been examined in detail. Here, we showed that miR-199a was downregulated in RCC and associated with poor prognostic phenotype. Using luciferase and western blot assays we identified that Rho-associated coiled coil-containing protein kinases 1 (ROCK1) was a direct target gene for miR-199a. miR-199a regulated proliferation, invasion, and apoptosis of clear cell renal cell carcinoma (ccRCC) cells by modulating ROCK1 expression. Interestingly, we also found that miR-199a was modulated by snail in ccRCC cells. Snail elevated ROCK1 expression by repressing miR-199a activity. Altogether, our results identify a crucial tumor suppressive role of miR-199a in the progression of ccRCC and suggest that miR-199a might be an anticancer therapeutic target for ccRCC patients.

  7. Role of Regulators of G Protein Signaling Proteins in Bone Physiology and Pathophysiology.

    Science.gov (United States)

    Jules, Joel; Yang, Shuying; Chen, Wei; Li, Yi-Ping

    2015-01-01

    Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins' regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases. © 2015 Elsevier Inc. All rights reserved.

  8. The STUbL RNF4 regulates protein group SUMOylation by targeting the SUMO conjugation machinery.

    Science.gov (United States)

    Kumar, Ramesh; González-Prieto, Román; Xiao, Zhenyu; Verlaan-de Vries, Matty; Vertegaal, Alfred C O

    2017-11-27

    SUMO-targeted ubiquitin ligases (STUbLs) mediate the ubiquitylation of SUMOylated proteins to modulate their functions. In search of direct targets for the STUbL RNF4, we have developed TULIP (targets for ubiquitin ligases identified by proteomics) to covalently trap targets for ubiquitin E3 ligases. TULIP methodology could be widely employed to delineate E3 substrate wiring. Here we report that the single SUMO E2 Ubc9 and the SUMO E3 ligases PIAS1, PIAS2, PIAS3, ZNF451, and NSMCE2 are direct RNF4 targets. We confirm PIAS1 as a key RNF4 substrate. Furthermore, we establish the ubiquitin E3 ligase BARD1, a tumor suppressor and partner of BRCA1, as an indirect RNF4 target, regulated by PIAS1. Interestingly, accumulation of BARD1 at local sites of DNA damage increases upon knockdown of RNF4. Combined, we provide an insight into the role of the STUbL RNF4 to balance the role of SUMO signaling by directly targeting Ubc9 and SUMO E3 ligases.

  9. Cellular senescence and tumor suppressor gene p16.

    Science.gov (United States)

    Rayess, Hani; Wang, Marilene B; Srivatsan, Eri S

    2012-04-15

    Cellular senescence is an irreversible arrest of cell growth. Biochemical and morphological changes occur during cellular senescence, including the formation of a unique cellular morphology such as flattened cytoplasm. Function of mitochondria, endoplasmic reticulum and lysosomes are affected resulting in the inhibition of lysosomal and proteosomal pathways. Cellular senescence can be triggered by a number of factors including, aging, DNA damage, oncogene activation and oxidative stress. While the molecular mechanism of senescence involves p16 and p53 tumor suppressor genes and telomere shortening, this review is focused on the mechanism of p16 control. The p16-mediated senescence acts through the retinoblastoma (Rb) pathway inhibiting the action of the cyclin dependant kinases leading to G1 cell cycle arrest. Rb is maintained in a hypophosphorylated state resulting in the inhibition of transcription factor E2F1. Regulation of p16 expression is complex and involves epigenetic control and multiple transcription factors. PRC1 (Pombe repressor complex (1) and PRC2 (Pombe repressor complex (2) proteins and histone deacetylases play an important role in the promoter hypermethylation for suppressing p16 expression. While transcription factors YY1 and Id1 suppress p16 expression, transcription factors CTCF, Sp1 and Ets family members activate p16 transcription. Senescence occurs with the inactivation of suppressor elements leading to the enhanced expression of p16. Copyright © 2011 UICC.

  10. Regulation and involvement in cancer and pathological conditions of MAGI1, a tight junction protein.

    Science.gov (United States)

    Feng, Xuemin; Jia, Shuqin; Martin, Tracey A; Jiang, Wen G

    2014-07-01

    Membrane-associated guanylate kinase with an inverted repeat member 1 (MAGI1), is a member of a family of proteins which are emerging as important in coupling the extracellular environment to intracellular signaling pathways and the cytoskeleton at synapses and tight junctions. Early studies described it as a scaffold protein localized at cell-cell junctions. Recently, MAGI1 was found to recruit various kinds of molecules via its PSD-95/Disks Large/Zonula Occludins (PDZ) domains to strengthen the junctional complex. There is an increasing body of evidence showing its involvement in receptor synaptic localization and the homeostasis of ion channels in the nervous system. Furthermore, evidence has accumulated to confirm the critical role of MAGI1 in regulating cell-cell contacts, which is always disrupted in tumor progression and is associated with invasiveness and metastasis. It has also been shown in vitro that the abnormal expression of MAGI1 influences the adhesion and invasiveness of cancer cells. Due to the presence of docking domains for PDZ-binding molecules, MAGI1 associates with a variety of molecules such as phosphatase and tensin homolog deleted on chromosome ten (PTEN), brain-specific angiogenesis inhibitor-1, β-catenin and the mouse homologue of the human NET1 DH domain protein. Pathway signaling analysis has indicated that MAGI1 is probably involved in many kinds of pathways especially the PTEN/Phosphatidylinositol-3 kinase/Akt pathway and the (Wg-Int)-β-catenin pathway which mediates intracellular functions. MAGI1 may therefore be a tumor suppressor and a therapy target for cancer and other diseases, although more in vitro and in vivo investigations are required. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. SpoVG Is a Conserved RNA-Binding Protein That Regulates Listeria monocytogenes Lysozyme Resistance, Virulence, and Swarming Motility.

    Science.gov (United States)

    Burke, Thomas P; Portnoy, Daniel A

    2016-04-05

    In this study, we sought to characterize the targets of the abundant Listeria monocytogenes noncoding RNA Rli31, which is required for L. monocytogenes lysozyme resistance and pathogenesis. Whole-genome sequencing of lysozyme-resistant suppressor strains identified loss-of-expression mutations in the promoter of spoVG , and deletion of spoVG rescued lysozyme sensitivity and attenuation in vivo of the rli31 mutant. SpoVG was demonstrated to be an RNA-binding protein that interacted with Rli31 in vitro . The relationship between Rli31 and SpoVG is multifaceted, as both the spoVG -encoded protein and the spoVG 5′-untranslated region interacted with Rli31. In addition, we observed that spoVG -deficient bacteria were nonmotile in soft agar and suppressor mutations that restored swarming motility were identified in the gene encoding a major RNase in Gram-positive bacteria, RNase J1. Collectively, these findings suggest that SpoVG is similar to global posttranscriptional regulators, a class of RNA-binding proteins that interact with noncoding RNA, regulate genes in concert with RNases, and control pleiotropic aspects of bacterial physiology. spoVG is widely conserved among bacteria; however, the function of this gene has remained unclear since its initial characterization in 1977. Mutation of spoVG impacts various phenotypes in Gram-positive bacteria, including methicillin resistance, capsule formation, and enzyme secretion in Staphylococcus aureus and also asymmetric cell division, hemolysin production, and sporulation in Bacillus subtilis . Here, we demonstrate that spoVG mutant strains of Listeria monocytogenes are hyper-lysozyme resistant, hypervirulent, nonmotile, and misregulate genes controlling carbon metabolism. Furthermore, we demonstrate that SpoVG is an RNA-binding protein. These findings suggest that SpoVG has a role in L. monocytogenes , and perhaps in other bacteria, as a global gene regulator. Posttranscriptional gene regulators help bacteria adapt to

  12. Glycolysis regulates the expansion of myeloid-derived suppressor cells in tumor-bearing hosts through prevention of ROS-mediated apoptosis

    Science.gov (United States)

    Jian, Shiou-Ling; Chen, Wei-Wei; Su, Yu-Chia; Su, Yu-Wen; Chuang, Tsung-Hsien; Hsu, Shu-Ching; Huang, Li-Rung

    2017-01-01

    Immunotherapy aiming to rescue or boost antitumor immunity is an emerging strategy for treatment of cancers. The efficacy of immunotherapy is strongly controlled by the immunological milieu of cancer patients. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cell populations with immunosuppressive functions accumulating in individuals during tumor progression. The signaling mechanisms of MDSC activation have been well studied. However, there is little known about the metabolic status of MDSCs and the physiological role of their metabolic reprogramming. In this study, we discovered that myeloid cells upregulated their glycolytic genes when encountered with tumor-derived factors. MDSCs exhibited higher glycolytic rate than their normal cell compartment did, which contributed to the accumulation of the MDSCs in tumor-bearing hosts. Upregulation of glycolysis prevented excess reactive oxygen species (ROS) production by MDSCs, which protected MDSCs from apoptosis. Most importantly, we identified the glycolytic metabolite, phosphoenolpyruvate (PEP), as a vital antioxidant agent able to prevent excess ROS production and therefore contributed to the survival of MDSCs. These findings suggest that glycolytic metabolites have important roles in the modulation of fitness of MDSCs and could be potential targets for anti-MDSC strategy. Targeting MDSCs with analogs of specific glycolytic metabolites, for example, 2-phosphoglycerate or PEP may diminish the accumulation of MDSCs and reverse the immunosuppressive milieu in tumor-bearing individuals. PMID:28492541

  13. Evaluating Modulators of ‘Regulator of G-protein Signaling’ (RGS) Proteins

    Science.gov (United States)

    Bosch, Dustin E.; Zielinski, Thomas; Lowery, Robert G.; Siderovski, David P.

    2012-01-01

    Regulator of G-protein Signaling’ (RGS) proteins constitute a class of intracellular signaling regulators that accelerate GTP hydrolysis by heterotrimeric Gα subunits. In recent years, RGS proteins have emerged as potential drug targets for small molecule modulation. Described in this unit are high-throughput screening procedures for identifying modulators of RGS protein-mediated GTPase acceleration (‘GAP activity’), for assessment of RGS domain/Gα interactions (most avid in vitro when Gα is bound by aluminum tetrafluoride), and for validation of candidate GAP-modulatory molecules with the single turnover GTP hydrolysis assay. PMID:22382998

  14. Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing

    DEFF Research Database (Denmark)

    Nagasawa, T; Zhang, Q; Raghunath, P N

    2006-01-01

    )-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma...... promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed...... differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target...

  15. Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

    Science.gov (United States)

    Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

    2016-01-01

    In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

  16. Protein import into plant mitochondria: signals, machinery, processing, and regulation.

    Science.gov (United States)

    Murcha, Monika W; Kmiec, Beata; Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F; Glaser, Elzbieta; Whelan, James

    2014-12-01

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

    Science.gov (United States)

    Labrecque, Mark P.; Takhar, Mandeep K.; Nason, Rebecca; Santacruz, Stephanie; Tam, Kevin J.; Massah, Shabnam; Haegert, Anne; Bell, Robert H.; Altamirano-Dimas, Manuel; Collins, Colin C.; Lee, Frank J.S.; Prefontaine, Gratien G.; Cox, Michael E.; Beischlag, Timothy V.

    2016-01-01

    Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. PMID:27015368

  18. Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species.

    Science.gov (United States)

    Kumar, Roshan; Bisht, Naveen C

    2018-02-01

    G-alpha (Gα) and 'Regulator of G-protein Signaling (RGS)' proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica 'U' triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.

  19. A conserved NAD+binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  20. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

    Directory of Open Access Journals (Sweden)

    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  1. Tumor suppressor gene p16/INK4A/CDKN2A-dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer.

    Science.gov (United States)

    Agarwal, Payal; Sandey, Maninder; DeInnocentes, Patricia; Bird, R Curtis

    2013-06-01

    p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16-defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16-transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re-feeding to induce cell cycle re-entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16-associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re-entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by (3)H-thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16-transfected CMT27A and CMT27H cells exited cell cycle post-serum-starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post-serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non-proliferative states. Copyright © 2012 Wiley Periodicals, Inc.

  2. Heat Shock Proteins in Tendinopathy: Novel Molecular Regulators

    Directory of Open Access Journals (Sweden)

    Neal L. Millar

    2012-01-01

    Full Text Available Tendon disorders—tendinopathies—are the primary reason for musculoskeletal consultation in primary care and account for up to 30% of rheumatological consultations. Whilst the molecular pathophysiology of tendinopathy remains difficult to interpret the disease process involving repetitive stress, and cellular load provides important mechanistic insight into the area of heat shock proteins which spans many disease processes in the autoimmune community. Heat shock proteins, also called damage-associated molecular patterns (DAMPs, are rapidly released following nonprogrammed cell death, are key effectors of the innate immune system, and critically restore homeostasis by promoting the reconstruction of the effected tissue. Our investigations have highlighted a key role for HSPs in tendion disease which may ultimately affect tissue rescue mechanisms in tendon pathology. This paper aims to provide an overview of the biology of heat shock proteins in soft tissue and how these mediators may be important regulators of inflammatory mediators and matrix regulation in tendinopathy.

  3. Heat Shock Proteins in Tendinopathy: Novel Molecular Regulators

    Science.gov (United States)

    Millar, Neal L.; Murrell, George A. C.

    2012-01-01

    Tendon disorders—tendinopathies—are the primary reason for musculoskeletal consultation in primary care and account for up to 30% of rheumatological consultations. Whilst the molecular pathophysiology of tendinopathy remains difficult to interpret the disease process involving repetitive stress, and cellular load provides important mechanistic insight into the area of heat shock proteins which spans many disease processes in the autoimmune community. Heat shock proteins, also called damage-associated molecular patterns (DAMPs), are rapidly released following nonprogrammed cell death, are key effectors of the innate immune system, and critically restore homeostasis by promoting the reconstruction of the effected tissue. Our investigations have highlighted a key role for HSPs in tendion disease which may ultimately affect tissue rescue mechanisms in tendon pathology. This paper aims to provide an overview of the biology of heat shock proteins in soft tissue and how these mediators may be important regulators of inflammatory mediators and matrix regulation in tendinopathy. PMID:23258952

  4. Regulation of thrombosis and vascular function by protein methionine oxidation

    Science.gov (United States)

    Gu, Sean X.; Stevens, Jeff W.

    2015-01-01

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  5. Interaction of 125I-labeled Ca2+-dependent regulator protein with cyclic nucleotide phosphodiesterase and its inhibitory protein

    International Nuclear Information System (INIS)

    Richman, P.G.; Klee, C.B.

    1978-01-01

    The Ca 2+ -dependent regulator protein of cyclic nucleotide phosphodiesterase was labeled with 125 I to the extent of 1 mol of monoiodotyrosine per mol. The iodinated protein showed a small decrease in affinity for phosphodiesterase but gave the same maximal level of activation of the enzyme as did the unmodified regulator protein. Iodinated regulator protein formed complexes with both highly purified cyclic nucleotide phosphodiesterase and phosphodiesterase inhibitory protein in the presence but not in the absence of Ca 2+ as demonstrated by ultracentrifugation in glycerol gradients. Cross-linking experiments indicate that the Ca 2+ -dependent regulator protein interacts with the large subunit of the inhibitory protein

  6. Regulation of Mdmx and its role in the p53 pathway

    NARCIS (Netherlands)

    Meulmeester, Erik

    2006-01-01

    The p53 protein is an important tumor suppressor that acts as a key regulator of the integrity of the genome. Two essential regulators of the p53 protein are Mdm2 and its homologue Mdmx. Like Mdm2, Mdmx represses p53-induced transcription. However, Mdmx cannot ubiquitinate or degrade p53 opposed to

  7. Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress.

    Science.gov (United States)

    Sieber, Jonas; Wieder, Nicolas; Ostrosky-Frid, Mauricio; Dvela-Levitt, Moran; Aygün, Ozan; Udeshi, Namrata D; Carr, Steven A; Greka, Anna

    2017-11-17

    The up-regulation of chaperones such as the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP or HSPA5) is part of the adaptive cellular response to endoplasmic reticulum (ER) stress. GRP78 is widely used as a marker of the unfolded protein response, associated with sustained ER stress. Here we report the discovery of a proteostatic mechanism involving GRP78 trimethylation in the context of ER stress. Using mass spectrometry-based proteomics, we identified two GRP78 fractions, one homeostatic and one induced by ER stress. ER stress leads to de novo biosynthesis of non-trimethylated GRP78, whereas homeostatic, METTL21A-dependent lysine 585-trimethylated GRP78 is reduced. This proteostatic mechanism, dependent on the posttranslational modification of GRP78, allows cells to differentially regulate specific protein abundance during cellular stress. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Glucose-regulated protein 94 deficiency induces squamous cell metaplasia and suppresses PTEN-null driven endometrial epithelial tumor development.

    Science.gov (United States)

    Shen, Jieli; Yao, Lijing; Lin, Yvonne G; DeMayo, Francesco J; Lydon, John P; Dubeau, Louis; Lee, Amy S

    2016-03-22

    Endometrial carcinoma is the most prevalent gynecologic cancer in the United States. The tumor suppressor gene Pten (phosphatase and tensin homolog) is commonly mutated in the more common type 1 (endometrioid) subtype. The glucose-regulated protein 94 (GRP94) is emerging as a novel regulator for cancer development. Here we report that expression profiles from the Cancer Genome Atlas (TCGA) showed significantly increased Grp94 mRNA levels in endometrial tumor versus normal tissues, correlating with highly elevated GRP94 protein expression in patient samples and the requirement of GRP94 for maintaining viability of human endometrioid adenocarcinoma (EAC) cell lines. Through generation of uterus-specific knockout mouse models with deletion of Grp94 alone (c94f/f) or in combination with Pten (cPf/f94f/f), we discovered that c94f/f uteri induced squamous cell metaplasia (SCM) and reduced active nuclear β-catenin. The cPf/f94f/f uteri showed accelerated SCM and suppression of PTEN-null driven EAC, with reduced cellular proliferation, attenuated β-catenin signaling and decreased AKT/S6 activation in the SCM. In contrast to single PTEN knockout uteri (cPf/f), cPf/f94f/f uteri showed no decrease in E-cadherin level and no invasive lesion. Collectively, our study implies that GRP94 downregulation induces SCM in EAC and suppresses AKT/S6 signaling, providing a novel mechanism for suppressing EAC progression.

  9. DNA polymerase eta is regulated by poly(rC)-binding protein 1 via mRNA stability

    Science.gov (United States)

    Ren, Cong; Cho, Seong-Jun; Jung, Yong-Sam; Chen, Xinbin

    2015-01-01

    DNA polymerase eta (POLH), a target of p53 tumor suppressor, plays a key role in translesion DNA synthesis (TLS). Loss of POLH is responsible for human cancer prone syndrome, Xeroderma Pigmentosum Variant (XPV). Due to its critical role in DNA repair and genome stability, POLH expression and activity are regulated by multiple pathways. In this study, we found that the levels of both POLH transcript and protein were decreased upon knockdown of the transcript encoding poly(rC)-binding protein 1 (PCBP1). We also found that the half-life of POLH mRNA was markedly decreased upon knockdown of PCBP1. Moreover, we found that PCBP1 directly bound to POLH 3′UTR and the PCBP1-binding site in POLH mRNA is an atypical AU-rich element. Finally, we showed that the AU-rich element in POLH 3′UTR was responsive to PCBP1 and sufficient for PCBP1 to regulate POLH expression. Altogether, we uncovered a novel mechanism by which POLH expression is controlled by PCBP1 via mRNA stability. PMID:25268038

  10. DNA polymerase η is regulated by poly(rC)-binding protein 1 via mRNA stability.

    Science.gov (United States)

    Ren, Cong; Cho, Seong-Jun; Jung, Yong-Sam; Chen, Xinbin

    2014-12-15

    POLH (DNA polymerase η), a target of p53 tumour suppressor, plays a key role in TLS (translesion DNA synthesis). Loss of POLH is responsible for the human cancer-prone syndrome XPV (xeroderma pigmentosum variant). Owing to its critical role in DNA repair and genome stability, POLH expression and activity are regulated by multiple pathways. In the present study, we found that the levels of both POLH transcript and protein were decreased upon knockdown of the transcript encoding PCBP1 [poly(rC)-binding protein 1]. We also found that the half-life of POLH mRNA was markedly decreased upon knockdown of PCBP1. Moreover, we found that PCBP1 directly bound to the POLH 3'-UTR and the PCBP1-binding site in POLH mRNA is an atypical AU-rich element. Finally, we showed that the AU-rich element in POLH 3'-UTR was responsive to PCBP1 and sufficient for PCBP1 to regulate POLH expression. Taken together, we uncovered a novel mechanism by which POLH expression is controlled by PCBP1 via mRNA stability.

  11. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  12. Eps15: a multifunctional adaptor protein regulating intracellular trafficking

    Directory of Open Access Journals (Sweden)

    van Bergen en Henegouwen Paul MP

    2009-10-01

    Full Text Available Abstract Over expression of receptor tyrosine kinases is responsible for the development of a wide variety of malignancies. Termination of growth factor signaling is primarily determined by the down regulation of active growth factor/receptor complexes. In recent years, considerable insight has been gained in the endocytosis and degradation of growth factor receptors. A crucial player in this process is the EGFR Protein tyrosine kinase Substrate #15, or Eps15. This protein functions as a scaffolding adaptor protein and is involved both in secretion and endocytosis. Eps15 has been shown to bind to AP-1 and AP-2 complexes, to bind to inositol lipids and to several other proteins involved in the regulation of intracellular trafficking. In addition, Eps15 has been detected in the nucleus of mammalian cells. Activation of growth factor receptors induces tyrosine phosphorylation and mono-ubiquitination of Eps15. The role of these post translational modifications of Eps15 is still a mystery. It is proposed that Eps15 and its family members Eps15R and Eps15b are involved in the regulation of membrane morphology, which is required for intracellular vesicle formation and trafficking.

  13. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    Science.gov (United States)

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

  14. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL

    DEFF Research Database (Denmark)

    Staller, Peter; Sulitkova, Jitka; Lisztwan, Joanna

    2003-01-01

    regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutation of the VHL...... of the CXCR4-specific ligand stromal cell-derived factor-1alpha (ref. 1). It is still uncertain how an evolving tumour cell is reprogrammed to express CXCR4, thus implementing the tendency to metastasize to specific organs. Here we show that the von Hippel-Lindau tumour suppressor protein pVHL negatively...

  15. Cauliflower mosaic virus protein P6 inhibits signaling responses to salicylic acid and regulates innate immunity.

    Directory of Open Access Journals (Sweden)

    Andrew J Love

    Full Text Available Cauliflower mosaic virus (CaMV encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA- and jasmonic acid (JA-dependent signaling and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst. Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants

  16. MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shouping [Department of Diagnostic Imaging, Linyi People' s Hospital, Linyi, Shandong 276000 (China); Wang, Xianjun [Department of Neurology, Linyi People' s Hospital, Linyi, Shandong 276000 (China); Li, Xiao [Department of Pathology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Cao, Yuandong, E-mail: yuandongcao@sina.com [Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province (China)

    2015-11-13

    MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays also revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. - Highlights: • miR-139-5p is downregulated in GBM. • miR-139-5p regulates cell proliferation through inducing apoptosis. • miR-139-5p regulates glioblastoma tumorigenesis by targeting 3′UTR of ELTD1. • miR-139-5p is involved in cell cycle regulation.

  17. The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins.

    Science.gov (United States)

    Höhne, Martin; Lorscheider, Johannes; von Bardeleben, Anna; Dufner, Matthias; Scharf, M Antonia; Gödel, Markus; Helmstädter, Martin; Schurek, Eva-Maria; Zank, Sibylle; Gerke, Peter; Kurschat, Christine; Sivritas, Sema Hayriye; Neumann-Haefelin, Elke; Huber, Tobias B; Reinhardt, H Christian; Schauss, Astrid C; Schermer, Bernhard; Fischbach, Karl-Friedrich; Benzing, Thomas

    2011-08-01

    Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.

  18. Tumor suppressor molecules and methods of use

    Science.gov (United States)

    Welch, Peter J.; Barber, Jack R.

    2004-09-07

    The invention provides substantially pure tumor suppressor nucleic acid molecules and tumor suppressor polypeptides. The invention also provides hairpin ribozymes and antibodies selective for these tumor suppressor molecules. Also provided are methods of detecting a neoplastic cell in a sample using detectable agents specific for the tumor suppressor nucleic acids and polypeptides.

  19. The Lin28/let-7 Axis Regulates Glucose Metabolism

    NARCIS (Netherlands)

    Zhu, Hao; Shyh-Chang, Ng; Segre, Ayellet V.; Shinoda, Gen; Shah, Samar P.; Einhorn, William S.; Takeuchi, Ayumu; Engreitz, Jesse M.; Hagan, John P.; Kharas, Michael G.; Urbach, Achia; Thornton, James E.; Triboulet, Robinson; Gregory, Richard I.; Altshuler, David; Daley, George Q.

    2011-01-01

    The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating-metabolism. When overexpressed in mice, both

  20. The von Hippel-Lindau (VHL) tumor-suppressor gene is down-regulated by selenium deficiency in Caco-2 cells and rat colon mucosa

    Science.gov (United States)

    To test the hypothesis that selenium affects DNA methylation and hence gene regulation we employed a methylation array (Panomics) in the human colonic epithelial Caco-2 cell model. The array profiles DNA methylation from promoter regions of 82 human genes. After conditioning cells to repeatedly redu...

  1. Tumor suppressor pten signaling is up-regulated in mammary epithelial cells by soy isoflavone genistein: implications for breast cancer protection

    Science.gov (United States)

    Epidemiological studies have shown lower occurrence of breast cancer in Asian women whose early intake of soy products is higher than their American counterparts. In a previous work, we showed protection against NMU-induced mammary tumors in rats exposed to dietary soy protein isolate (SPI) or casei...

  2. The role of protein phosphatase 2A in regulating Wnt signaling and apoptosis

    Science.gov (United States)

    Li, Xinghai

    Protein phosphatase 2A (PP2A) is a major serine/threonine-specific phosphatase and regulates a significant array of cellular events. This dissertation primarily describes the novel role of PP2A in Wnt signaling and apoptosis. First, PP2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus. PP2A is required for β- catenin degradation in vitro. A PP2A heterotrimer containing A, C, and B56 subunits was co- immunoprecipitated with axin. A, C, and B56 subunits each have ventralizing ability in Xenopus embryos. B56 was epistatically positioned downstream of GSK3β and axin but upstream of β-catenin. Second, B56-targeted PP2A is required for survival and protects from apoptosis in Drosophila. Loss of A, C, or B56 subunits by RNA interference (RNAi) induced apoptosis in S2 cells, which requires the presence of specific caspases. Epistasis analysis placed B56-targeted PP2A functionally upstream of Apaf-1, Reaper and Hid, and p53. Loss of B56-targeted PP2A in Drosophila embryos by RNAi resulted in abortion of embryo development and this phenotype was rescued by co-RNAi of Drice. Third, two conserved domains in B subunits mediate binding to the A subunit of PP2A. B subunits have no detectable sequence homology among different families. In vitro expression of a series of B56α fragments identified two distinct domains that independently bound to the A subunit. Sequence alignment of these A subunit-binding domains recognized conserved residues in B/PR55 and B'/PR72 family members that serve a similar function. Fourth, to examine whether the B56β gene within 11q12 is a tumor suppressor mutated in neuroblastoma, the DNA and RNA samples from neuroblastoma patients and cell lines were analyzed and no mutations were identified in the coding regions of the B56β gene. Finally, to identify novel regulatory subunits of PP2A in S. cerevisiae , biochemical approaches for purifying PP2A-associated novel regulators were undertaken. Although the A and C subunit complex in the

  3. Distinct phosphorylation events regulate p130- and p107-mediated repression of E2F-4

    DEFF Research Database (Denmark)

    Farkas, Thomas; Hansen, Klaus; Holm, Karin

    2002-01-01

    The "pocket proteins" pRb (retinoblastoma tumor suppressor protein), p107, and p130 regulate cell proliferation via phosphorylation-sensitive interactions with E2F transcription factors and other proteins. We previously identified 22 in vivo phosphorylation sites in human p130, including three...

  4. Dynamic protein complexes regulate NF-kappaB signaling.

    Science.gov (United States)

    Wegener, E; Krappmann, D

    2008-01-01

    NF-kappaB is a major regulator of the first-line defense against invading pathogens, antigen-specific adaptive immune responses or chemical stress. Stimulation either by extracellular ligands (e.g., inflammatory cytokines, microbial pathogens, peptide antigens) or by intracellular Stressors (e.g., genotoxic drugs) initiates signal-specific pathways that all converge at the IkappaB kinase (IKK) complex, the gatekeeper for NF-kappaB activation. During recent years, considerable progress has been made in understanding the function of NF-kappaB in the regulation of cell growth, survival and apoptosis. In this review, we will focus on the regulation of large signaling complexes on the route to NF-kappaB. Recently published data demonstrate that the assembly, maintenance and activity of the IKK complex determine downstream activation of NF-kappaB. In addition, dynamic complexes upstream of IKK are formed in response to tumor necrosis factor (TNF), antigenic peptides or DNA-damaging agents. Clustering of signaling adaptors promotes the association and activation of ubiquitin ligases that trigger the conjugation of regulatory ubiquitin to target proteins. Ubiquitination serves as a platform to recruit the IKK complex and potentially other protein kinases to trigger IKK activation. These findings support a concept whereby protein complex assembly induces regulatory ubiquitination, which in turn recruits and activates protein kinases. Notably, the great interest in a detailed description of the mechanisms that regulate NF-kappaB activity stems from many observations that link dysregulated NF-kappaB signaling with the onset or progression of various diseases, including cancer, chronic inflammation, cardiovascular disorders and neurodegenerative diseases. Thus, the formation of large signaling clusters and regulatory ubiquitin chains represents promising targets for pharmacological intervention to modulate NF-kappaB signal transduction in disease.

  5. RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

    Science.gov (United States)

    2010-06-01

    lysed in triple- detergent RIPA buffer with protease inhibitor cocktail (Roche, Pleasanton, CA). For nuclear and cytoplasmic fractionation, the NE-PER kit...Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR. The Journal of allergy and clinical immunology 2008, 121(4):853-859...and western blot analysis. Western analysis was performed as described previously.12 For detection of VEGFα and TSP1 from tumors, triple- detergent

  6. Emerging Roles of Regulators of G Protein Signaling (RGS) Proteins in the Immune System.

    Science.gov (United States)

    Druey, Kirk M

    2017-01-01

    The regulators of G protein signaling (RGS) proteins are a large, evolutionarily conserved group of intracellular proteins expressed in every cell type and tissue throughout the body including the immune system. Through their signature GTPase-activating protein (GAP) activity on heterotrimeric G proteins and interactions with signaling complexes and membrane constituents (e.g., lipids), RGS proteins determine the intensity and duration of G protein-coupled receptor-induced responses. They may also have a function in generating intracellular signaling gradients necessary for the directional migration of leukocytes to inflamed tissues containing local accumulations of chemoattractants. Although physiological functions of most RGS proteins in leukocytes and lymphoid organs are largely unknown, it appears thus far that deficiency of individual RGS proteins in mice does not affect homeostatic immune responses in the absence of immunogenic challenge and/or microbial infection. Although aberrant expression of some RGS proteins has been linked to dysregulated immunity and/or neoplasia in humans, there are no human diseases attributed to specific RGS dysfunction. Here, we highlight mostly published work describing expression and functions of the core group of RGS proteins that were among the first discovered, in both innate and adaptive immune processes, with particular emphasis on cell trafficking. © 2017 Elsevier Inc. All rights reserved.

  7. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    Science.gov (United States)

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  8. CHIP is a novel tumor suppressor in pancreatic cancer and inhibits tumor growth through targeting EGFR

    Science.gov (United States)

    Wang, Tianxiao; Yang, Jingxuan; Xu, Jianwei; Li, Jian; Cao, Zhe; Zhou, Li; You, Lei; Shu, Hong; Lu, Zhaohui; Li, Huihua; Li, Min; Zhang, Taiping; Zhao, Yupei

    2014-01-01

    Carboxyl terminus of heat shock protein 70-interacting protein (CHIP) is an E3 ubiquitin ligase that is involved in protein quality control and mediates several tumor-related proteins in many cancers, but the function of CHIP in pancreatic cancer is not known. Here we show that CHIP interacts and ubiquitinates epidermal growth factor receptor (EGFR) for proteasome-mediated degradation in pancreatic cancer cells, thereby inhibiting the activation of EGFR downstream pathways. CHIP suppressed cell proliferation, anchor-independent growth, invasion and migration, as well as enhanced apoptosis induced by erlotinib in vitro and in vivo. The expression of CHIP was decreased in pancreatic cancer tissues or sera. Low CHIP expression in tumor tissues was correlated with tumor differentiation and shorter overall survival. These observations indicate that CHIP serves as a novel tumor suppressor by down-regulating EGFR pathway in pancreatic cancer cells, decreased expression of CHIP was associated with poor prognosis in pancreatic cancer. PMID:24722501

  9. Chromatin-regulating proteins as targets for cancer therapy

    International Nuclear Information System (INIS)

    Oike, Takahiro; Ogiwara, Hideaki; Kohno, Takashi; Amornwichet, Napapat; Nakano, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. (author)

  10. Forkhead Box Protein A2 (FOXA2) Protein Stability and Activity Are Regulated by Sumoylation

    OpenAIRE

    Belaguli, Narasimhaswamy S.; Zhang, Mao; Brunicardi, F. Charles; Berger, David H.

    2012-01-01

    The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 t...

  11. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

    2010-01-01

    The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT...... cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been...

  12. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis.

    Science.gov (United States)

    Kamp, Marjon E; Liu, Youtao; Kortholt, Arjan

    2016-01-14

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis.

  13. Heat Shock Protein 90 regulates encystation in Entamoeba

    Directory of Open Access Journals (Sweden)

    Meetali eSingh

    2015-10-01

    Full Text Available Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90 in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

  14. Heat Shock Protein 90 regulates encystation in Entamoeba

    Science.gov (United States)

    Singh, Meetali; Sharma, Shalini; Bhattacharya, Alok; Tatu, Utpal

    2015-01-01

    Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90) in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania, and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba. PMID:26528271

  15. MiR-424 Promotes Non-Small Cell Lung Cancer Progression and Metastasis through Regulating the Tumor Suppressor Gene TNFAIP1

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2017-05-01

    Full Text Available Background/Aims: This study aimed to investigate the potential roles of miR-424 expression in non-small cell lung cancer (NSCLC metastasis and growth and its underlying mechanism. Methods: The expression of miR-424 in two NSCLC cell lines (A549 and H1975 was altered by transfection with miR-424 mimic and inhibitor. Effects of miR-424 overexpression and suppression on cells migration, invasion and colony formation were analyzed. Target genes for miR-424 were predicted using bioinformatics method and then verified using luciferase assay. Effects of miR-424 expression on cell migration, invasion and proliferation were reanalyzed on the condition of TNFAIP1 was silenced. Moreover, TNFAIP1 silencing and miR-424 modified A549 cells were subcutaneous injected into node BALB/c mice to confirm the regulation of miR-424 on TNFAIP1 in regulating tumor growth. Results: Compared with the control, miR-424 overexpression significantly increased the migrated and invaded cells, as well as the proliferated colonies. TNFAIP1 was a predicted target gene for miR-424, and was negatively regulated by miR-424. TNFAIP1 silence significantly increased the migrated and invaded cells compared to that in control, while these increases were abolished by miR-424 suppression. Animal experiment further evidenced miR-424 affected tumor growth by regulating TNFAIP1. Conclusions: These data demonstrate that miR-424 may be a contributor for NSCLC progression and metastasis through involving in cell migration, invasion and proliferation via inhibiting TNFAIP1. This study may provide theoretical basis for miR-424 in NSCLC target therapeutic treatment.

  16. Dual-specificity tyrosine-regulated kinase 2 is a suppressor and potential prognostic marker for liver metastasis of colorectal cancer.

    Science.gov (United States)

    Ito, Daisuke; Yogosawa, Satomi; Mimoto, Rei; Hirooka, Shinichi; Horiuchi, Takashi; Eto, Ken; Yanaga, Katsuhiko; Yoshida, Kiyotsugu

    2017-08-01

    Colorectal cancer is a common cancer and a leading cause of cancer-related death worldwide. The liver is a dominant metastatic site for patients with colorectal cancer. Molecular mechanisms that allow colorectal cancer cells to form liver metastases are largely unknown. Activation of epithelial-mesenchymal transition is the key step for metastasis of cancer cells. We recently reported that dual-specificity tyrosine-regulated kinase 2 (DYRK2) controls epithelial-mesenchymal transition in breast cancer and ovarian serous adenocarcinoma. The aim of this study is to clarify whether DYRK2 regulates liver metastases of colorectal cancer. We show that the ability of cell invasion and migration was abrogated in DYRK2-overexpressing cells. In an in vivo xenograft model, liver metastatic lesions were markedly diminished by ectopic expression of DYRK2. Furthermore, we found that patients whose liver metastases expressed low DYRK2 levels had significantly worse overall and disease-free survival. Given the findings that DYRK2 regulates cancer cell metastasis, we concluded that the expression status of DYRK2 could be a predictive marker for liver metastases of colorectal cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  17. Tumor-derived exosomes induce CD8+T cell suppressors.

    Science.gov (United States)

    Maybruck, Brian T; Pfannenstiel, Lukas W; Diaz-Montero, Marcela; Gastman, Brian R

    2017-08-15

    The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8 + T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8 + T cells are tumor-derived exosomes (TDEs). Membrane vesicles and TDEs from multiple head and neck cancer cell line's conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3 + CD8 + T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8 + T cells followed by suppression analysis. Using fractionated conditioned growth media, factors >200 kDa induced CD8 + T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8 + T cells. For the first-time, TDEs have been identified to induce a

  18. Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH.

    Science.gov (United States)

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang; Chang, Wen

    2012-04-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

  19. Modulation of allogeneic stimulation in man. I. Characterization of an in vitro induced suppressor macrophage population

    International Nuclear Information System (INIS)

    Stux, S.V.; Dubey, D.P.; Yunis, E.J.

    1981-01-01

    Cultured human peripheral blood mononuclear cells suppressed the allogeneic response of fresh autologous lymphocytes. This suppressor activity developed gradually over a period of one week. The cells primarily responsible for this effect were enriched by Ficoll density gradient centrifugation. It was found that the suppressor cell is a large, low density nylon wool adherent, radioresistant, phagocytic, and nonspecific esterase positive mononuclear cell. Moreover, these cells did not form E rosettes and were Fc positive. Electron microscopy confirmed that suppressor cells were macrophage like. Suppressor activity was not due to cytotoxicity, crowding, or steric hinderance by the cultured cells. The suppressor macrophage population did not appear to inhibit the allogeneic response via prostaglandin or arginase release, or interfere with the tritiated thymidine uptake by release of endogenous thymidine. The above system is viewed as an in vitro model of immune regulation by suppressor macrophages, in the context of allogeneic response

  20. Forkhead box protein A2 (FOXA2 protein stability and activity are regulated by sumoylation.

    Directory of Open Access Journals (Sweden)

    Narasimhaswamy S Belaguli

    Full Text Available The forkhead box protein A2 (FOXA2 is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1 and mapped the sumoylation site to the amino acid lysine 6 (K6. Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  1. Forkhead box protein A2 (FOXA2) protein stability and activity are regulated by sumoylation.

    Science.gov (United States)

    Belaguli, Narasimhaswamy S; Zhang, Mao; Brunicardi, F Charles; Berger, David H

    2012-01-01

    The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on Pdx-1 area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.

  2. p53 regulates expression of uncoupling protein 1 through binding and repression of PPARγ coactivator-1α

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Fjære, Even; Liaset, Bjørn

    2016-01-01

    The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP...

  3. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  4. Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

    Science.gov (United States)

    Farhy Tselnicker, Isabella; Tsemakhovich, Vladimir; Rishal, Ida; Kahanovitch, Uri; Dessauer, Carmen W; Dascal, Nathan

    2014-04-01

    Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li(+) did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gβγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

  5. Scalloped a member of the Hippo tumor suppressor pathway controls mushroom body size in Drosophila brain by non-canonical regulation of neuroblast proliferation.

    Science.gov (United States)

    Rohith, Basavanahalli Nanjundaiah; Shyamala, Baragur Venkatanarayanasetty

    2017-12-15

    Cell proliferation, growth and survival are three different basic processes which converge at determining a fundamental property -the size of an organism. Scalloped (Sd) is the first characterised transcriptional partner to Yorkie (Yki), the downstream effector of the Hippo pathway which is a highly potential and evolutionarily conserved regulator of organ size. Here we have studied the hypomorphic effect of sd on the development of Mushroom Bodies (MBs) in Drosophila brain. We show that, sd non-function results in an increase in the size of MBs. We demonstrate that, sd regulation on MB size operates through multiple routes. Sd expressed in the differentiated MB neurons, imposes non-cell autonomous repression on the proliferation of MB precursor cells, and Sd expression in the MB neuroblasts (NB) cell autonomously represses mushroom body neuroblast (MBNB) proliferation. Further Sd in Kenyon cells (KCs) imparts a cell autonomous restriction on their growth. Our findings are distinctive because, while the classical sd loss of function phenotypes in eye, wing and lymph gland are reported as loss of tissue or reduced organ size, the present study shows that, Sd inactivation in the developing MB, promotes precursor cell proliferation and results in an increase in the organ size. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    Science.gov (United States)

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

  7. Regulation of protein synthesis during sea urchin early development

    Energy Technology Data Exchange (ETDEWEB)

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  8. Redox Signaling Regulated by Cysteine Persulfide and Protein Polysulfidation.

    Science.gov (United States)

    Kasamatsu, Shingo; Nishimura, Akira; Morita, Masanobu; Matsunaga, Tetsuro; Abdul Hamid, Hisyam; Akaike, Takaaki

    2016-12-15

    For decades, reactive persulfide species including cysteine persulfide (CysSSH) have been known to exist endogenously in organisms. However, the physiological significance of endogenous persulfides remains poorly understood. That cystathionine β-synthase and cystathionine γ-lyase produced CysSSH from cystine was recently demonstrated. An endogenous sulfur transfer system involving CysSSH evidently generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo. Because reactive persulfide species such as CysSSH and GSSH have higher nucleophilicity than parental cysteine (Cys) and glutathione do, these reactive species exhibit strong scavenging activities against oxidants, e.g., hydrogen peroxide, and electrophiles, which contributes to redox signaling regulation. Also, several papers indicated that various proteins and enzymes have Cys polysulfides including CysSSH at their specific Cys residues, which is called protein polysulfidation. Apart from the redox signaling regulatory mechanism, another plausible function of protein polysulfidation is providing protection for protein thiol residues against irreversible chemical modification caused by oxidants and electrophiles. Elucidation of the redox signaling regulatory mechanism of reactive persulfide species including small thiol molecules and thiol-containing proteins should lead to the development of new therapeutic strategies and drug discoveries for oxidative and electrophilic stress-related diseases.

  9. The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Sofie eVan Rossom

    2012-07-01

    Full Text Available DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine-nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2 to 6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human HEK293T cells. Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.

  10. Phosphorylation of transcriptional regulators in the retinoblastoma protein pathway by UL97, the viral cyclin-dependent kinase encoded by human cytomegalovirus.

    Science.gov (United States)

    Iwahori, Satoko; Kalejta, Robert F

    2017-12-01

    Human cytomegalovirus (HCMV) encodes a viral cyclin-dependent kinase (v-CDK), the UL97 protein. UL97 phosphorylates Rb, p107 and p130, thereby inactivating all three retinoblastoma (Rb) family members. Rb proteins function through regulating the activity of transcription factors to which they bind. Therefore, we examined whether the UL97-mediated regulation of the Rb tumor suppressors also extended to their binding partners. We observed that UL97 phosphorylates LIN52, a component of p107- and p130-assembled transcriptionally repressive DREAM complexes that control transcription during the G0/G1 phases, and the Rb-associated E2F3 protein that activates transcription through G1 and S phases. Intriguingly, we also identified FoxM1B, a transcriptional regulator during the S and G2 phases, as a UL97 substrate. This survey extends the influence of UL97 beyond simply the Rb proteins themselves to their binding partners, as well as past the G1/S transition into later stages of the cell cycle. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

    International Nuclear Information System (INIS)

    Yu Dehua; Fan, Wufang; Liu, Guohong; Nguy, Vivian; Chatterton, Jon E.; Long Shilong; Ke, Ning; Meyhack, Bernd; Bruengger, Adrian; Brachat, Arndt; Wong-Staal, Flossie; Li, Qi-Xiang

    2006-01-01

    HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties

  12. Deciphering the BRCA1 Tumor Suppressor Network*

    Science.gov (United States)

    Jiang, Qinqin; Greenberg, Roger A.

    2015-01-01

    The BRCA1 tumor suppressor protein is a central constituent of several distinct macromolecular protein complexes that execute homology-directed DNA damage repair and cell cycle checkpoints. Recent years have borne witness to an exciting phase of discovery at the basic molecular level for how this network of DNA repair proteins acts to maintain genome stability and suppress cancer. The clinical dividends of this investment are now being realized with the approval of first-in-class BRCA-targeted therapies for ovarian cancer and identification of molecular events that determine responsiveness to these agents. Further delineation of the basic science underlying BRCA network function holds promise to maximally exploit genome instability for hereditary and sporadic cancer therapy. PMID:26048987

  13. Extravirgin olive oil up-regulates CB₁ tumor suppressor gene in human colon cancer cells and in rat colon via epigenetic mechanisms.

    Science.gov (United States)

    Di Francesco, Andrea; Falconi, Anastasia; Di Germanio, Clara; Micioni Di Bonaventura, Maria Vittoria; Costa, Antonio; Caramuta, Stefano; Del Carlo, Michele; Compagnone, Dario; Dainese, Enrico; Cifani, Carlo; Maccarrone, Mauro; D'Addario, Claudio

    2015-03-01

    Extravirgin olive oil (EVOO) represents the typical lipid source of the Mediterranean diet, an eating habit pattern that has been associated with a significant reduction of cancer risk. Diet is the more studied environmental factor in epigenetics, and many evidences suggest dysregulation of epigenetic pathways in cancer. The aim of our study was to investigate the effects of EVOO and its phenolic compounds on endocannabinoid system (ECS) gene expression via epigenetic regulation in both human colon cancer cells (Caco-2) and rats exposed to short- and long-term dietary EVOO. We observed a selective and transient up-regulation of CNR1 gene - encoding for type 1 cannabinoid receptor (CB₁) - that was evoked by exposure of Caco-2 cells to EVOO (100 ppm), its phenolic extracts (OPE, 50 μM) or authentic hydroxytyrosol (HT, 50 μM) for 24 h. None of the other major elements of the ECS (i.e., CB₂; GPR55 and TRPV1 receptors; and NAPE-PLD, DAGL, FAAH and MAGL enzymes) was affected at any time point. The stimulatory effect of OPE and HT on CB₁ expression was inversely correlated to DNA methylation at CNR1 promoter and was associated with reduced proliferation of Caco-2 cells. Interestingly, CNR1 gene was less expressed in Caco-2 cells when compared to normal colon mucosa cells, and again this effect was associated with higher level of DNA methylation at CNR1. Moreover, in agreement with the in vitro studies, we also observed a remarkable (~4-fold) and selective increase in CB₁ expression in the colon of rats receiving dietary EVOO supplementation for 10 days. Consistently, CpG methylation of rat Cnr1 promoter, miR23a and miR-301a, previously shown to be involved in the pathogenesis of colorectal cancer and predicted to target CB₁ mRNA, was reduced after EVOO administration down to ~50% of controls. Taken together, our findings demonstrating CB₁ gene expression modulation by EVOO or its phenolic compounds via epigenetic mechanism, both in vitro and in vivo, may

  14. DMPD: Post-transcriptional regulation of proinflammatory proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15075353 Post-transcriptional regulation of proinflammatory proteins. Anderson P, P...l) (.csml) Show Post-transcriptional regulation of proinflammatory proteins. PubmedID 15075353 Title Post-tr...anscriptional regulation of proinflammatory proteins. Authors Anderson P, Phillip

  15. Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

    Directory of Open Access Journals (Sweden)

    Eduardo Cebollero

    2012-01-01

    Full Text Available During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

  16. Membrane Association of the PTEN Tumor Suppressor: Molecular Details of the Protein-Membrane Complex from SPR Binding Studies and Neutron Reflection

    Science.gov (United States)

    Shenoy, Siddharth; Shekhar, Prabhanshu; Heinrich, Frank; Daou, Marie-Claire; Gericke, Arne; Ross, Alonzo H.; Lösche, Mathias

    2012-01-01

    The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 µM and 0.4 µM, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. PMID:22505997

  17. The BRCA1 Tumor Suppressor Binds to Inositol 1,4,5-Trisphosphate Receptors to Stimulate Apoptotic Calcium Release*

    Science.gov (United States)

    Hedgepeth, Serena C.; Garcia, M. Iveth; Wagner, Larry E.; Rodriguez, Ana M.; Chintapalli, Sree V.; Snyder, Russell R.; Hankins, Gary D. V.; Henderson, Beric R.; Brodie, Kirsty M.; Yule, David I.; van Rossum, Damian B.; Boehning, Darren

    2015-01-01

    The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein. PMID:25645916

  18. FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice.

    Science.gov (United States)

    Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J

    2017-04-11

    The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk), an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis). We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. FAD Regulates CRYPTOCHROME Protein Stability and Circadian Clock in Mice

    Directory of Open Access Journals (Sweden)

    Arisa Hirano

    2017-04-01

    Full Text Available The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD binding pocket of CRYPTOCHROME2 (CRY2, a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin kinase (Rfk, an FAD biosynthetic enzyme, enhanced CRY degradation. RFK protein levels and FAD concentrations oscillate in the nucleus, suggesting that they are subject to circadian control. Knockdown of Rfk combined with a riboflavin-deficient diet altered the CRY levels in mouse liver and the expression profiles of clock and clock-controlled genes (especially those related to metabolism including glucose homeostasis. We conclude that light-independent mechanisms of FAD regulate CRY and contribute to proper circadian oscillation of metabolic genes in mammals.

  20. Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

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    Guanggan Hu

    2007-03-01

    Full Text Available A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP-dependent protein kinase A (PKA is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential

  1. FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-β/Smad2/3 signaling pathway.

    Science.gov (United States)

    Shi, Jie-Yi; Ma, Li-Jie; Zhang, Ji-Wei; Duan, Meng; Ding, Zhen-Bin; Yang, Liu-Xiao; Cao, Ya; Zhou, Jian; Fan, Jia; Zhang, Xiaoming; Zhao, Ying-Jun; Wang, Xiao-Ying; Gao, Qiang

    2017-09-13

    FOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC). Expression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing. FOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-β/Smad2/3 signaling pathway. Our findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-β/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.

  2. A Dictyostelium chalone uses G proteins to regulate proliferation

    Directory of Open Access Journals (Sweden)

    Hanson Nana E

    2009-07-01

    Full Text Available Abstract Background Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. Results We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA. Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus, whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. Conclusion This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  3. A Dictyostelium chalone uses G proteins to regulate proliferation.

    Science.gov (United States)

    Bakthavatsalam, Deenadayalan; Choe, Jonathan M; Hanson, Nana E; Gomer, Richard H

    2009-07-27

    Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  4. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    Science.gov (United States)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  5. Suppressors of RNA silencing encoded by tomato leaf curl

    Indian Academy of Sciences (India)

    Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B ... In the present study suppressor function of ORF C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl ...

  6. Regulation of the pancreatic duodenal homeobox-1 protein by DNA-dependent protein kinase.

    Science.gov (United States)

    Lebrun, Patricia; Montminy, Marc R; Van Obberghen, Emmanuel

    2005-11-18

    The transcription factor PDX-1 plays a crucial role during pancreatic development and in the function of insulin-producing beta cells. Disruption of the pdx-1 gene in these cells induces overt diabetes in mice, and this gene is modified in several type 2 diabetic families. It is thus crucial to determine the molecular mechanisms involved in the regulation of PDX-1 expression and/or activation. We identified new proteins associated with PDX-1 by mass spectrometry. These proteins, Ku70 and Ku80, are regulatory subunits of DNA-dependent protein kinase (DNA-PK). We determined that the interaction between PDX-1 and Ku70 or Ku80 is dependent on the homeodomain of PDX-1. Most interestingly, we demonstrated in vitro that the DNA-PK phosphorylates PDX-1 on threonine 11. Although this residue is located in the transactivation domain, this phosphorylation does not seem to be implicated in the transcriptional activation of PDX-1. However, in response to radiation, which activates DNA-PK, a second form of the PDX-1 protein appears rapidly. This form is phosphorylated on threonine and seems to drive PDX-1 degradation by the proteosome. In correlation with this degradation, we observed a subsequent reduction in the activation of the insulin promoter and a decrease in PDX-1-mediated gene expression, i.e. glut2 and glucokinase. Our study demonstrates that radiation, through the activation of DNA-PK, may regulate PDX-1 protein expression.

  7. Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

    Science.gov (United States)

    Si, Yuan; Ji, Xinyan; Cao, Xiaolei; Dai, Xiaoming; Xu, Lingyi; Zhao, Hongxia; Guo, Xiaocan; Yan, Huan; Zhang, Haitao; Zhu, Chu; Zhou, Qi; Tang, Mei; Xia, Zongping; Li, Li; Cong, Yu-Sheng; Ye, Sheng; Liang, Tingbo; Feng, Xin-Hua; Zhao, Bin

    2017-09-15

    The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR . ©2017 American Association for Cancer Research.

  8. R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity.

    Science.gov (United States)

    Xie, Zhihui; Chan, Eunice C; Druey, Kirk M

    2016-03-01

    G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily--the focus of this review--includes RGS1-5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.

  9. Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

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    Zhenyong Wu

    2017-10-01

    Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

  10. Bunched, the Drosophila homolog of the mammalian tumor suppressor TSC-22, promotes cellular growth

    Directory of Open Access Journals (Sweden)

    Wu Xiaodong

    2008-01-01

    Full Text Available Abstract Background Transforming Growth Factor-β1 stimulated clone-22 (TSC-22 is assumed to act as a negative growth regulator and tumor suppressor. TSC-22 belongs to a family of putative transcription factors encoded by four distinct loci in mammals. Possible redundancy among the members of the TSC-22/Dip/Bun protein family complicates a genetic analysis. In Drosophila, all proteins homologous to the TSC-22/Dip/Bun family members are derived from a single locus called bunched (bun. Results We have identified bun in an unbiased genetic screen for growth regulators in Drosophila. Rather unexpectedly, bun mutations result in a growth deficit. Under standard conditions, only the long protein isoform BunA – but not the short isoforms BunB and BunC – is essential and affects growth. Whereas reducing bunA function diminishes cell number and cell size, overexpression of the short isoforms BunB and BunC antagonizes bunA function. Conclusion Our findings establish a growth-promoting function of Drosophila BunA. Since the published studies on mammalian systems have largely neglected the long TSC-22 protein version, we hypothesize that the long TSC-22 protein is a functional homolog of BunA in growth regulation, and that it is antagonized by the short TSC-22 protein.

  11. Tumor Suppressor Function of CYLD in Nonmelanoma Skin Cancer

    Directory of Open Access Journals (Sweden)

    K. C. Masoumi

    2011-01-01

    Full Text Available Ubiquitin and ubiquitin-related proteins posttranslationally modify substrates, and thereby alter the functions of their targets. The ubiquitination process is involved in various physiological responses, and dysregulation of components of the ubiquitin system has been linked to many diseases including skin cancer. The ubiquitin pathways activated among skin cancers are highly diverse and may reflect the various characteristics of the cancer type. Basal cell carcinoma and squamous cell carcinoma, the most common types of human skin cancer, are instances where the involvement of the deubiquitination enzyme CYLD has been recently highlighted. In basal cell carcinoma, the tumor suppressor protein CYLD is repressed at the transcriptional levels through hedgehog signaling pathway. Downregulation of CYLD in basal cell carcinoma was also shown to interfere with TrkC expression and signaling, thereby promoting cancer progression. By contrast, the level of CYLD is unchanged in squamous cell carcinoma, instead, catalytic inactivation of CYLD in the skin has been linked to the development of squamous cell carcinoma. This paper will focus on the current knowledge that links CYLD to nonmelanoma skin cancers and will explore recent insights regarding CYLD regulation of NF-κB and hedgehog signaling during the development and progression of these types of human tumors.

  12. ARS2 is a general suppressor of pervasive transcription.

    Science.gov (United States)

    Iasillo, Claudia; Schmid, Manfred; Yahia, Yousra; Maqbool, Muhammad A; Descostes, Nicolas; Karadoulama, Evdoxia; Bertrand, Edouard; Andrau, Jean-Christophe; Jensen, Torben Heick

    2017-09-29

    Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II at protein-coding (pc) genes. Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication-dependent histone-, promoter upstream transcript- and enhancer RNA-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate pc gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  14. Regulation of G Protein-Coupled Receptors by Ubiquitination

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    Kamila Skieterska

    2017-04-01

    Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

  15. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  16. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  17. PTEN: a yin-yang master regulator protein in health and disease.

    Science.gov (United States)

    Pulido, Rafael

    2015-05-01

    The PTEN gene is a tumor suppressor gene frequently mutated in human tumors, which encodes a ubiquitous protein whose major activity is to act as a lipid phosphatase that counteracts the action of the oncogenic PI3K. In addition, PTEN displays protein phosphatase- and catalytically-independent activities. The physiologic control of PTEN function, and its inactivation in cancer and other human diseases, including some neurodevelopmental disorders, is upon the action of multiple regulatory mechanisms. This provides a wide spectrum of potential therapeutic approaches to reconstitute PTEN activity. By contrast, inhibition of PTEN function may be beneficial in a different group of human diseases, such as type 2 diabetes or neuroregeneration-related pathologies. This makes PTEN a functionally dual yin-yang protein with high potential in the clinics. Here, a brief overview on PTEN and its relation with human disease is presented. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Overexpression of Exportin-5 Overrides the Inhibitory Effect of miRNAs Regulation Control and Stabilize Proteins via Posttranslation Modifications in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Naseruddin Höti

    2017-10-01

    Full Text Available Although XPO5 has been characterized to have tumor-suppressor features in the miRNA biogenesis pathway, the impact of altered expression of XPO5 in cancers is unexplored. Here we report a novel “oncogenic” role of XPO5 in advanced prostate cancer. Using prostate cancer models, we found that excess levels of XPO5 override the inhibitory effect of the canoncial miRNA-mRNA regulation, resulting in a global increase in proteins expression. Importantly, we found that decreased expression of XPO5 could promote an increase in proteasome degradation, whereas overexpression of XPO5 leads to altered protein posttranslational modification via hyperglycosylation, resulting in cellular protein stability. We evaluated the therapeutic advantage of targeting XPO5 in prostate cancer and found that knocking down XPO5 in prostate cancer cells suppressed cellular proliferation and tumor development without significantly impacting normal fibroblast cells survival. To our knowledge, this is the first report describing the oncogenic role of XPO5 in overriding the miRNAs regulation control. Furthermore, we believe that these findings will provide an explanation as to why, in some cancers that express higher abundance of mature miRNAs, fail to suppress their potential protein targets.

  19. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

    Science.gov (United States)

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-05-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation.

  20. Leptin and Fasting Regulate Rat Gastric Glucose-Regulated Protein 58

    Directory of Open Access Journals (Sweden)

    Susana B. Bravo

    2011-01-01

    Full Text Available The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58 is a molecular chaperone member of the endoplasmic reticulum (ER stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency. We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.

  1. Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

    Directory of Open Access Journals (Sweden)

    José Luis Carrasco

    Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

  2. Structure of the Tetrameric p53 Tumor Suppressor Bound to DNA

    National Research Council Canada - National Science Library

    Marmorstein, Ronen

    2002-01-01

    The p53 tumor suppressor binds DNA as a tetramer to regulate the transcription of genes involved in cell cycle arrest and apoptosis, and alterations in the DNA-binding core domain of p53 are the most...

  3. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA

    Directory of Open Access Journals (Sweden)

    Russomano Fabio B

    2008-10-01

    Full Text Available Abstract Background The oncoproteins of human papillomavirus (HPVs directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. Methods We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA and correlated this to HIV-1 and HPV infection. Results HIV/HPV co-infection was associated with a significant increase in expression (p 2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9. Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005, in comparison to controls. Conclusion Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development.

  4. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA)

    International Nuclear Information System (INIS)

    Nicol, Alcina F; Pirmez, Claude; Pires, Andréa Rodrigues Cordovil; Souza, Simone R de; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Silva, José R Lapa e

    2008-01-01

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm 2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm 2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development

  5. Cell-cycle and suppressor proteins expression in uterine cervix in HIV/HPV co-infection: comparative study by tissue micro-array (TMA).

    Science.gov (United States)

    Nicol, Alcina F; Pires, Andréa Rodrigues Cordovil; de Souza, Simone R; Nuovo, Gerard J; Grinsztejn, Beatriz; Tristão, Aparecida; Russomano, Fabio B; Velasque, Luciane; Lapa e Silva, José R; Pirmez, Claude

    2008-10-07

    The oncoproteins of human papillomavirus (HPVs) directly effect cell-cycle control. We hypothesize that regulatory and cell cycle protein expression might be additionally modified in the cervix of HIV/HPV co-infected women. We analyzed the expression of Rb, p27, VEGF and Elf-1 transcriptor factor by immunohistochemistry in 163 paraffin-embeded cervical samples using Tissue Micro-Array (TMA) and correlated this to HIV-1 and HPV infection. HIV/HPV co-infection was associated with a significant increase in expression (p < 0.001) of VEGF and p27 in both low and high grade CIN when compared to the cervices of women infected by HPV alone. Decreased Rb expression was evident with increased CIN grade in the cervices of women infected with HPV alone (p = 0.003 average of cells/mm2 in CIN I: 17.9, CIN II/III: 4.8, and tumor 3.9). Rb expression increased 3-fold for both low and high grade CIN with HPV/HIV-1 co-infection compared to HPV infection alone but did not reach statistical significance. There was a significant increase in Elf-1 expression in HPV+/HIV- women with CIN II/III and tumor (average of cells/mm2 in CIN I: 63.8; CIN II/III: 115.7 and tumor: 112.0, p = 0.005), in comparison to controls. Co-infection of HPV and HIV leads to significant increase in the VEGF and p27 expression when compared to HPV+/HIV-negative infection that could facilitate viral persistence and invasive tumor development.

  6. Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean.

    Science.gov (United States)

    Choudhury, Swarup Roy; Pandey, Sona

    2015-11-01

    Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants. © 2015 American Society of Plant Biologists. All rights reserved.

  7. The protein tyrosine kinase Tec regulates mast cell function.

    Science.gov (United States)

    Schmidt, Uwe; Abramova, Anastasia; Boucheron, Nicole; Eckelhart, Eva; Schebesta, Alexandra; Bilic, Ivan; Kneidinger, Michael; Unger, Bernd; Hammer, Martina; Sibilia, Maria; Valent, Peter; Ellmeier, Wilfried

    2009-11-01

    Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

  8. A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

    Science.gov (United States)

    Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H; Sobieski, Courtney; Zhou, Hao; Konopko, Aaron M; Martin, Brent R; Mennerick, Steven J; Blumer, Kendall J

    2014-02-28

    Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

  9. Long term effect of curcumin in restoration of tumour suppressor p53 and phase-II antioxidant enzymes via activation of Nrf2 signalling and modulation of inflammation in prevention of cancer.

    Directory of Open Access Journals (Sweden)

    Laxmidhar Das

    Full Text Available Inhibition of carcinogenesis may be a consequence of attenuation of oxidative stress via activation of antioxidant defence system, restoration and stabilization of tumour suppressor proteins along with modulation of inflammatory mediators. Previously we have delineated significant role of curcumin during its long term effect in regulation of glycolytic pathway and angiogenesis, which in turn results in prevention of cancer via modulation of stress activated genes. Present study was designed to investigate long term effect of curcumin in regulation of Nrf2 mediated phase-II antioxidant enzymes, tumour suppressor p53 and inflammation under oxidative tumour microenvironment in liver of T-cell lymphoma bearing mice. Inhibition of Nrf2 signalling observed during lymphoma progression, resulted in down regulation of phase II antioxidant enzymes, p53 as well as activation of inflammatory signals. Curcumin potentiated significant increase in Nrf2 activation. It restored activity of phase-II antioxidant enzymes like GST, GR, NQO1, and tumour suppressor p53 level. In addition, curcumin modulated inflammation via upregulation of TGF-β and reciprocal regulation of iNOS and COX2. The study suggests that during long term effect, curcumin leads to prevention of cancer by inducing phase-II antioxidant enzymes via activation of Nrf2 signalling, restoration of tumour suppressor p53 and modulation of inflammatory mediators like iNOS and COX2 in liver of lymphoma bearing mice.

  10. Ion channel regulation by protein S-acylation

    Science.gov (United States)

    2014-01-01

    Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease. PMID:24821965

  11. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

    Science.gov (United States)

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-01-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  12. Quantitative proteomics reveals differential regulation of protein expression in recipient myocardium after trilineage cardiovascular cell transplantation

    Science.gov (United States)

    Chang, Ying-Hua; Ye, Lei; Cai, Wenxuan; Lee, Yoonkyu; Guner, Huseyin; Lee, Youngsook; Kamp, Timothy J.; Zhang, Jianyi; Ge, Ying

    2015-01-01

    Intramyocardial transplantation of cardiomyocytes (CMs), endothelial cells (ECs), and smooth muscle cells (SMCs) derived from human induced pluripotent stem cells (hiPSCs) has beneficial effects on the post-infarction heart. However, the mechanisms underlying the functional improvements remain undefined. We employed large-scale label-free quantitative proteomics to identify proteins that were differentially regulated following cellular transplantation in a swine model of myocardial infarction (MI). We identified 22 proteins that were significantly up-regulated after trilineage cell transplantation compared to both MI and Sham groups. Among them, 12 proteins, including adenylyl cyclase-associated protein 1 and tropomodulin-1, are associated with positive regulation of muscular contraction whereas 11 proteins, such as desmoplakin and zyxin, are involved in embryonic and muscular development and regeneration. Moreover, we identified 21 proteins up-regulated and another 21 down-regulated in MI, but reversed after trilineage cell transplantation. Proteins up-regulated after MI but reversed by transplantation are related to fibrosis and apoptosis. Conversely, proteins down-regulated in MI but restored after cell therapy are regulators of protein nitrosylation. Our results show that the functionally beneficial effects of trilineage cell therapy are accompanied by differential regulation of protein expression in the recipient myocardium, which may contribute to the improved cardiac function. PMID:26033914

  13. Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles.

    Science.gov (United States)

    Adams, Katherine R; Chauhan, Sitara; Patel, Divya B; Clements, Virginia K; Wang, Yan; Jay, Steven M; Edwards, Nathan J; Ostrand-Rosenberg, Suzanne; Fenselau, Catherine

    2018-01-05

    Ubiquitinated proteins carried by the extracellular vesicles (EV) released by myeloid-derived suppressor cells (MDSC) have been investigated using proteomic strategies to examine the effect of tumor-associated inflammation. EV were collected from MDSC directly following isolation from tumor-bearing mice with low and high inflammation. Among the 1092 proteins (high inflammation) and 925 proteins (low inflammation) identified, more than 50% were observed as ubiquitinated proteoforms. More than three ubiquitin-attachment sites were characterized per ubiquitinated protein, on average. Multiple ubiquitination sites were identified in the pro-inflammatory proteins S100 A8 and S100 A9, characteristic of MDSC and in histones and transcription regulators among other proteins. Spectral counting and pathway analysis suggest that ubiquitination occurs independently of inflammation. Some ubiquitinated proteins were shown to cause the migration of MDSC, which has been previously connected with immune suppression and tumor progression. Finally, MDSC EV are found collectively to carry all the enzymes required to catalyze ubiquitination, and the hypothesis is presented that a portion of the ubiquitinated proteins are produced in situ.

  14. Potential of lactic acid bacteria as suppressors of wine allergies

    Directory of Open Access Journals (Sweden)

    Yıldırım Hatice Kalkan

    2017-01-01

    Full Text Available Allergens causes some symptoms as all asthma, allergic conjunctivitis, and allergic rhinitis. These symptoms are seen twice as many in women than in men. The major wine allergens reported in wines are endochitinase 4A and lipid-transfer protein (LTP. This review deal with possibilities of using lactic acid bacteria as suppressors of wine allergies. Phenolic compounds present in wines have not only antioxidant properties causing radical scavenging but also some special properties reported in many in vitro studies as regulating functions in inflammatory cells as mast cells. So what is the role of lactic acid bacteria in these cases? Lactic acid bacteria are used during malolactic fermentation step of wine production with purpose of malic acid reduction. During this bioconversion complex phenolic compounds could be hydrolysed by bacterial enzymes to their aglycone forms. Obtained aglycons could pass through the intestinal epithelium of human and allowed reduction of IgE antibody production by affecting Th1/ Th2 ratio. Considering different contents and quantities of phenols in different grape varieties and consequently in different wines more studies are required in order to determine which lactic acid bacteria and strains could be effective in suppressing wine allergens.

  15. Drafting the proteome landscape of myeloid-derived suppressor cells.

    Science.gov (United States)

    Gato, María; Blanco-Luquin, Idoia; Zudaire, Maribel; de Morentin, Xabier Martínez; Perez-Valderrama, Estela; Zabaleta, Aintzane; Kochan, Grazyna; Escors, David; Fernandez-Irigoyen, Joaquín; Santamaría, Enrique

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that are defined by their myeloid origin, immature state, and ability to potently suppress T-cell responses. They regulate immune responses and the population significantly increases in the tumor microenvironment of patients with glioma and other malignant tumors. For their study, MDSCs are usually isolated from the spleen or directly of tumors from a large number of tumor-bearing mice although promising ex vivo differentiated MDSC production systems have been recently developed. During the last years, proteomics has emerged as a powerful approach to analyze MDSCs proteomes using shotgun-based mass spectrometry (MS), providing functional information about cellular homeostasis and metabolic state at a global level. Here, we will revise recent proteome profiling studies performed in MDSCs from different origins. Moreover, we will perform an integrative functional analysis of the protein compilation derived from these large-scale proteomic studies in order to obtain a comprehensive view of MDSCs biology. Finally, we will also discuss the potential application of high-throughput proteomic approaches to study global proteome dynamics and post-translational modifications (PTMs) during the differentiation process of MDSCs that will greatly boost the identification of novel MDSC-specific therapeutic targets to apply in cancer immunotherapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. SIRT3: Oncogene and Tumor Suppressor in Cancer

    Directory of Open Access Journals (Sweden)

    Margalida Torrens-Mas

    2017-07-01

    Full Text Available Sirtuin 3 (SIRT3, the major deacetylase in mitochondria, plays a crucial role in modulating oxygen reactive species (ROS and limiting the oxidative damage in cellular components. SIRT3 targets different enzymes which regulate mitochondrial metabolism and participate in ROS detoxification, such as the complexes of the respiratory chain, the isocitrate dehydrogenase, or the manganese superoxide dismutase. Thus, SIRT3 activity is essential in maintaining mitochondria homeostasis and has recently received great attention, as it is considered a fidelity protein for mitochondrial function. In some types of cancer, SIRT3 functions as a tumoral promoter, since it keeps ROS levels under a certain threshold compatible with cell viability and proliferation. On the contrary, other studies describe SIRT3 as a tumoral suppressor, as SIRT3 could trigger cell death under stress conditions. Thus, SIRT3 could have a dual role in cancer. In this regard, modulation of SIRT3 activity could be a new target to develop more personalized therapies against cancer.

  17. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  18. Analysis of the Yeast Kinome Reveals a Network of Regulated Protein Localization during Filamentous Growth

    OpenAIRE

    Bharucha, Nikë; Ma, Jun; Dobry, Craig J.; Lawson, Sarah K.; Yang, Zhifen; Kumar, Anuj

    2008-01-01

    The subcellular distribution of kinases and other signaling proteins is regulated in response to cellular cues; however, the extent of this regulation has not been investigated for any gene set in any organism. Here, we present a systematic analysis of protein kinases in the budding yeast, screening for differential localization during filamentous growth. Filamentous growth is an important stress response involving mitogen-activated protein kinase and cAMP-dependent protein kinase signaling m...

  19. The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici.

    Science.gov (United States)

    Eichmann, Ruth; Schultheiss, Holger; Kogel, Karl-Heinz; Hückelhoven, Ralph

    2004-05-01

    BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.

  20. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

    Science.gov (United States)

    Kovács, Krisztián A; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J; Cardinaux, Jean-René

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  2. Needleless emulsion electrospinning for the regulated delivery of susceptible proteins.

    Science.gov (United States)

    Buzgo, Matej; Filova, Eva; Staffa, Andrea Mickova; Rampichova, Michala; Doupnik, Miroslav; Vocetkova, Karolina; Lukasova, Vera; Kolcun, Radka; Lukas, David; Necas, Alois; Amler, Evzen

    2018-03-01

    In the present work, we developed a novel needleless emulsion electrospinning technique that improves the production rate of the core/shell production process. The nanofibres are based on poly-ε-caprolactone (PCL) as a continuous phase combined with a droplet phase based on Pluronic F-68 (PF-68). The PCL-PF-68 nanofibres show a time-regulated release of active molecules. Needleless emulsion electrospinning was used to encapsulate a diverse set of compounds to the core phase [i.e. 5-(4,6-dichlorotriazinyl) aminofluorescein -PF-68, horseradish peroxidase, Tetramethylrhodamine-dextran, insulin growth factor-I, transforming growth factor-β and basic fibroblast growth factor]. In addition, the PF-68 facilitates the preservation of the bioactivity of delivered proteins. The system's potential was highlighted by an improvement in the metabolic activity and proliferation of mesenchymal stem cells. The developed system has the potential to deliver susceptible molecules in tissue-engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Regulation of PCNA-protein interactions for genome stability

    DEFF Research Database (Denmark)

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...

  4. The effects of calcium regulation of endosperm reserve protein ...

    African Journals Online (AJOL)

    The effects of steep liquor calcium ion on sorghum endosperm reserve protein mobilization were evaluated using two improved Nigeria sorghum cultivars (ICSV 400 and KSV 8). The key protein modification factors evaluated were free amino nitrogen (FAN), total non protein nitrogen (TNPN) and soluble protein of cold water ...

  5. The Fanconi anemia proteins functionally interact with the protein kinase regulated by RNA (PKR).

    Science.gov (United States)

    Zhang, Xiaoling; Li, June; Sejas, Daniel P; Rathbun, Keaney R; Bagby, Grover C; Pang, Qishen

    2004-10-15

    Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

  6. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  7. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  8. The evolution of regulators of G protein signalling proteins as drug targets - 20 years in the making: IUPHAR Review 21.

    Science.gov (United States)

    Sjögren, B

    2017-03-01

    Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets. © 2017 The British Pharmacological Society.

  9. Remodeling epigenetic modifications at tumor suppressor gene promoters with bovine oocyte extract.

    Science.gov (United States)

    Wang, Zhenfei; Yue, Yongli; Han, Pengyong; Sa, Rula; Ren, Xiaolv; Wang, Jie; Bai, Haidong; Yu, Haiquan

    2013-09-01

    Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and histone modifications at their promoter regions plays an important role in the initiation and progression of cancer. The therapeutic effect of the widely used epigenetic drugs, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, remains unsatisfactory. One important underlying factor in the ineffectiveness of these drugs is that their actions lack specificity. To investigate whether oocyte extract can be used for epigenetic re-programming of cancer cells, H460 human lung cancer cells were reversibly permeabilized and incubated with bovine oocyte extract. Bisulfite sequencing showed that bovine oocyte extract induced significant demethylation at hypermethylated promoter CpG islands of the tumor suppressor genes RUNX3 and CDH1; however, the DNA methylation levels of repetitive sequences were not affected. Chromatin immunoprecipitation showed that bovine oocyte extract significantly reduced transcriptionally repressive histone modifications and increased transcriptionally activating histone modifications at the promoter regions of RUNX3 and CDH1. Bovine oocyte extract reactivated the expression of RUNX3 and CDH1 at both the messenger RNA and the protein levels without up-regulating the transcription of pluripotency-associated genes. At the functional level, anchorage-independent proliferation, migration and invasion of H460 cells was strongly inhibited. These results demonstrate that bovine oocyte extract reactivates epigenetically silenced tumor suppressor genes by remodeling the epigenetic modifications at their promoter regions. Bovine oocyte extract may provide a useful tool for investigating epigenetic mechanisms in cancer and a valuable source for developing novel safe therapeutic approaches that target epigenetic alterations. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

    DEFF Research Database (Denmark)

    Mannervik, M; Fan, S; Ström, A C

    1999-01-01

    of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation......Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression...... during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription...

  11. Noise suppressor for turbo fan jet engines

    Science.gov (United States)

    Cheng, D. Y. (Inventor)

    1983-01-01

    A noise suppressor is disclosed for installation on the discharge or aft end of a turbo fan engine. Within the suppressor are fixed annular airfoils which are positioned to reduce the relative velocity between the high temperature fast moving jet exhaust and the low temperature slow moving air surrounding it. Within the suppressor nacelle is an exhaust jet nozzle which constrains the shape of the jet exhaust to a substantially uniform elongate shape irrespective of the power setting of the engine. Fixed ring airfoils within the suppressor nacelle therefore have the same salutary effects irrespective of the power setting at which the engine is operated.

  12. Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

    Science.gov (United States)

    Doveston, Richard G; Kuusk, Ave; Andrei, Sebastian A; Leysen, Seppe; Cao, Qing; Castaldi, Maria P; Hendricks, Adam; Brunsveld, Luc; Chen, Hongming; Boyd, Helen; Ottmann, Christian

    2017-08-01

    14-3-3 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs. © 2017 Federation of European Biochemical Societies.

  13. The DNA damage repair protein Ku70 interacts with FOXO4 to coordinate a conserved cellular stress response

    NARCIS (Netherlands)

    A.B. Brenkman (Arjan); N.J.F. van den Broek (Niels); P.L.J. de Keizer (Peter); D.C. van Gent (Dik); B.M. Burgering (Boudewijn)

    2010-01-01

    textabstractIn this study, we searched for proteins regulating the tumor suppressor and life-span regulator FOXO4. Through an unbiased tandem-affinity purification strategy combined with mass spectrometry, we identified the heterodimer Ku70/Ku80 (Ku), a DNA double-strand break repair component.

  14. Suppressors made from intermetallic materials

    Science.gov (United States)

    Klett, James W; Muth, Thomas R; Cler, Dan L

    2014-11-04

    Disclosed are several examples of apparatuses for suppressing the blast and flash produced as a projectile is expelled by gases from a firearm. In some examples, gases are diverted away from the central chamber to an expansion chamber by baffles. The gases are absorbed by the expansion chamber and desorbed slowly, thus decreasing pressure and increasing residence time of the gases. In other examples, the gases impinge against a plurality of rods before expanding through passages between the rods to decrease the pressure and increase the residence time of the gases. These and other exemplary suppressors are made from an intermetallic material composition for enhanced strength and oxidation resistance at high operational temperatures.

  15. Regulation of dynamin family proteins by post-translational ...

    Indian Academy of Sciences (India)

    Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agentsinvolved in several biological processes such as endocytosis, maintenance of organelle morphology and viralresistance. These large GTPases couple GTP hydrolysis with membrane alterations such as ...

  16. Identification of C/EBPα as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anna M Marthaler

    2017-06-01

    Full Text Available Patients suffering from Epidermodysplasia verruciformis (EV, a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV, such as HPV type 8. They have a high risk to develop non-melanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203, a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein α (C/EBPα, a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBPα/mir-203-pathway. As a consequence, the miR-203 target ΔNp63α, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBPα but not C/EBPβ expression at the transcriptional level. As shown in knock-down experiments, C/EBPα is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBPα regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBPα and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBPα/mir-203-dependent

  17. Synthetic lethal interaction between the tumour suppressor STAG2 and its paralog STAG1.

    Science.gov (United States)

    Benedetti, Lorena; Cereda, Matteo; Monteverde, LeeAnn; Desai, Nikita; Ciccarelli, Francesca D

    2017-06-06

    Cohesin is a multi-protein complex that tethers sister chromatids during mitosis and mediates DNA repair, genome compartmentalisation and regulation of gene expression. Cohesin subunits frequently acquire cancer loss-of-function alterations and act as tumour suppressors in several tumour types. This has led to increased interest in cohesin as potential target in anti-cancer therapy. Here we show that the loss-of-function of STAG2, a core component of cohesin and an emerging tumour suppressor, leads to synthetic dependency of mutated cancer cells on its paralog STAG1. STAG1 and STAG2 share high sequence identity, encode mutually exclusive cohesin subunits and retain partially overlapping functions. We inhibited STAG1 and STAG2 in several cancer cell lines where the two genes have variable mutation and copy number status. In all cases, we observed that the simultaneous blocking of STAG1 and STAG2 significantly reduces cell proliferation. We further confirmed the synthetic lethal interaction developing a vector-free CRISPR system to induce STAG1/STAG2 double gene knockout. We provide strong evidence that STAG1 is a promising therapeutic target in cancers with inactivating alterations of STAG2.

  18. Coordinating ERK signaling via the molecular scaffold Kinase Suppressor of Ras [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Danielle Frodyma

    2017-08-01

    Full Text Available Many cancers, including those of the colon, lung, and pancreas, depend upon the signaling pathways induced by mutated and constitutively active Ras. The molecular scaffolds Kinase Suppressor of Ras 1 and 2 (KSR1 and KSR2 play potent roles in promoting Ras-mediated signaling through the Raf/MEK/ERK kinase cascade. Here we summarize the canonical role of KSR in cells, including its central role as a scaffold protein for the Raf/MEK/ERK kinase cascade, its regulation of various cellular pathways mediated through different binding partners, and the phenotypic consequences of KSR1 or KSR2 genetic inactivation. Mammalian KSR proteins have a demonstrated role in cellular and organismal energy balance with implications for cancer and obesity. Targeting KSR1 in cancer using small molecule inhibitors has potential for therapy with reduced toxicity to the patient. RNAi and small molecule screens using KSR1 as a reference standard have the potential to expose and target vulnerabilities in cancer. Interestingly, although KSR1 and KSR2 are similar in structure, KSR2 has a distinct physiological role in regulating energy balance. Although KSR proteins have been studied for two decades, additional analysis is required to elucidate both the regulation of these molecular scaffolds and their potent effect on the spatial and temporal control of ERK activation in health and disease.

  19. Direct regulation of transforming growth factor β-induced epithelial-mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells.

    Science.gov (United States)

    Kusunose, Masaaki; Hashimoto, Naozumi; Kimura, Motohiro; Ogata, Ryo; Aoyama, Daisuke; Sakamoto, Koji; Miyazaki, Shinichi; Ando, Akira; Omote, Norihito; Imaizumi, Kazuyoshi; Kawabe, Tsutomu; Hasegawa, Yoshinori

    2015-12-01

    Transforming growth factor β (TGFβ) causes the acquisition of epithelial-mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ-induced phosphorylation of its C-terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C-terminus (PTEN4A), but not PTEN wild, inhibits TGFβ-induced EMT. Nevertheless, whether or not the blockade of TGFβ-induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C-terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ-induced EMT in lung cancer cells. The unphosphorylated PTEN C-terminus might not directly retain the phosphatase activities and repress TGFβ-induced EMT; the modification that keeps the PTEN C-terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ-induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C-terminus might be a therapeutic target to negatively regulate TGFβ-induced EMT in lung cancer cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  20. Positive muscle protein net balance and differential regulation of atrogene expression after resistance exercise and milk protein supplementation

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2014-01-01

    Purpose Resistance exercise and amino acid availability are positive regulators of muscle protein net balance (NB). However, anabolic responses to resistance exercise and protein supplementation deserve further elucidation. The purpose was to compare intakes of whey, caseinate (both: 0.30 g/kg lean...

  1. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  2. Cellular prion protein expression is not regulated by the Alzheimer's amyloid precursor protein intracellular domain.

    Directory of Open Access Journals (Sweden)

    Victoria Lewis

    Full Text Available There is increasing evidence of molecular and cellular links between Alzheimer's disease (AD and prion diseases. The cellular prion protein, PrP(C, modulates the post-translational processing of the AD amyloid precursor protein (APP, through its inhibition of the β-secretase BACE1, and oligomers of amyloid-β bind to PrP(C which may mediate amyloid-β neurotoxicity. In addition, the APP intracellular domain (AICD, which acts as a transcriptional regulator, has been reported to control the expression of PrP(C. Through the use of transgenic mice, cell culture models and manipulation of APP expression and processing, this study aimed to clarify the role of AICD in regulating PrP(C. Over-expression of the three major isoforms of human APP (APP(695, APP(751 and APP(770 in cultured neuronal and non-neuronal cells had no effect on the level of endogenous PrP(C. Furthermore, analysis of brain tissue from transgenic mice over-expressing either wild type or familial AD associated mutant human APP revealed unaltered PrP(C levels. Knockdown of endogenous APP expression in cells by siRNA or inhibition of γ-secretase activity also had no effect on PrP(C levels. Overall, we did not detect any significant difference in the expression of PrP(C in any of the cell or animal-based paradigms considered, indicating that the control of cellular PrP(C levels by AICD is not as straightforward as previously suggested.

  3. Regulation of Polysaccharide- and Protein- Specific Antibody Responses to Intact Extracellular Bacteria

    Science.gov (United States)

    2016-03-11

    1 | P a g e “Regulation of polysaccharide- and protein- specific antibody responses to intact extracellular bacteria ” By Swagata Kar...the thesis manuscript entitled: “Regulation of polysaccharide- and protein- specific antibody responses to intact extracellular bacteria ” Is...polysaccharide- and protein- specific antibody responses to intact extracellular bacteria ” Author: Swagata Kar, Ph.D., 2016. Uniformed Services University

  4. Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

    KAUST Repository

    Altawashi, Azza

    2012-02-28

    Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation. 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Regulation of protein homeostasis in neurodegenerative diseases : the role of coding and non-coding genes

    NARCIS (Netherlands)

    Alvarenga Fernandes Sin, Olga; Nollen, Ellen A. A.

    2015-01-01

    Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding,

  6. FAD regulates CRYPTOCHROME protein stability and circadian clock in mice

    OpenAIRE

    Hirano, Arisa; Braas, Daniel; Fu, Ying-Hui; Ptáček, Louis J.

    2017-01-01

    The circadian clock generates biological rhythms of metabolic and physiological processes, including the sleep-wake cycle. We previously identified a missense mutation in the flavin adenine dinucleotide (FAD) binding pocket of CRYPTOCHROME2 (CRY2), a clock protein that causes human advanced sleep phase. This prompted us to examine the role of FAD as a mediator of the clock and metabolism. FAD stabilized CRY proteins, leading to increased protein levels. In contrast, knockdown of Riboflavin ki...

  7. Proteomic evaluation and validation of cathepsin D regulated proteins in macrophages exposed to Streptococcus pneumoniae.

    Science.gov (United States)

    Bewley, Martin A; Pham, Trong K; Marriott, Helen M; Noirel, Josselin; Chu, Hseuh-Ping; Ow, Saw Y; Ryazanov, Alexey G; Read, Robert C; Whyte, Moira K B; Chain, Benny; Wright, Phillip C; Dockrell, David H

    2011-06-01

    Macrophages are central effectors of innate immune responses to bacteria. We have investigated how activation of the abundant macrophage lysosomal protease, cathepsin D, regulates the macrophage proteome during killing of Streptococcus pneumoniae. Using the cathepsin D inhibitor pepstatin A, we demonstrate that cathepsin D differentially regulates multiple targets out of 679 proteins identified and quantified by eight-plex isobaric tag for relative and absolute quantitation. Our statistical analysis identified 18 differentially expressed proteins that passed all paired t-tests (α = 0.05). This dataset was enriched for proteins regulating the mitochondrial pathway of apoptosis or inhibiting competing death programs. Five proteins were selected for further analysis. Western blotting, followed by pharmacological inhibition or genetic manipulation of cathepsin D, verified cathepsin D-dependent regulation of these proteins, after exposure to S. pneumoniae. Superoxide dismutase-2 up-regulation was temporally related to increased reactive oxygen species generation. Gelsolin, a known regulator of mitochondrial outer membrane permeabilization, was down-regulated in association with cytochrome c release from mitochondria. Eukaryotic elongation factor (eEF2), a regulator of protein translation, was also down-regulated by cathepsin D. Using absence of the negative regulator of eEF2, eEF2 kinase, we confirm that eEF2 function is required to maintain expression of the anti-apoptotic protein Mcl-1, delaying macrophage apoptosis and confirm using a murine model that maintaining eEF2 function is associated with impaired macrophage apoptosis-associated killing of Streptococcus pneumoniae. These findings demonstrate that cathepsin D regulates multiple proteins controlling the mitochondrial pathway of macrophage apoptosis or competing death processes, facilitating intracellular bacterial killing.

  8. Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

    Science.gov (United States)

    Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

    2017-10-01

    Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  9. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    International Nuclear Information System (INIS)

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-01-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with ( 32 P) PO 4 3- and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone

  10. Expression and Location of Glucose-regulated Protein 78 in Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    W Wang

    2014-04-01

    Full Text Available Objective: To know the role of glucose-regulated protein 78 (GRP78/BiP/HSPA5 in spermatogenesis and its expression and location in the testis and epididymis. Methods: Immunohistochemistry and Western blot were used to detect GRP78 location and expression in the testis and epididymis. Results: Glucose-regulated protein 78 was observed in spermatocytes, round spermatids and interstitial cells of the testis and in principal cells of the epididymis. Glucose-regulated protein 78 was first detected in the rat testis at postnatal day 14. Thereafter, the protein level increased gradually with age and was maintained at a high and stable state after postnatal day 28. In the rat, GRP78 was expressed in the principal cells but not in clear cells of the epididymis. Conclusion: Glucose-regulated protein 78 participates in the process of spermatogenesis.

  11. Regulation of dynamin family proteins by post-translational ...

    Indian Academy of Sciences (India)

    2017-04-22

    Apr 22, 2017 ... Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane ...

  12. The effects of calcium regulation of endosperm reserve protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-15

    Jun 15, 2011 ... according to Holmes (1991) modification of the recommended methods of analysis # 25 (1986). Soluble protein of cold water extract (CWS-protein) was measured using a modification of. Coomassie brilliant blue method of Lewis et al. (1979) as described by Holmes (1991) using bovine serum albumen as ...

  13. Fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75 interact with the CREC proteins, calumenin and reticulocalbin

    DEFF Research Database (Denmark)

    Hansen, Gry Aune Westergaard; Ludvigsen, Maja; Jacobsen, Christian

    2015-01-01

    Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted...

  14. Protein kinase A regulates AKAP250 (gravin) scaffold binding to the β2-adrenergic receptor

    OpenAIRE

    Tao, Jiangchuan; Wang, Hsien-yu; Malbon, Craig C.

    2003-01-01

    A-kinase-anchoring protein 250 (AKAP250; gravin) acts as a scaffold that binds protein kinase A (PKA), protein kinase C and protein phosphatases, associating reversibly with the β2-adrenergic receptor. The receptor-binding domain of the scaffold and the regulation of the receptor–scaffold association was revealed through mutagenesis and biochemical analyses. The AKAP domain found in other members of this superfamily is essential for the scaffold–receptor interactions. Gravin constructs lackin...

  15. Characterisation of components and mechanisms involved in redox-regulation of protein import into chloroplasts

    OpenAIRE

    Stengel, Anna

    2009-01-01

    The vast majority of chloroplast proteins is encoded in the nucleus and thus has to be posttranslationally imported into the organelle, a process that is facilitated by two multimeric protein machineries, the Toc and Tic complexes (translocon at the outer/inner envelope of chloroplasts). Regulation of protein import, e.g. by redox signals, is a crucial step to adapt the protein content to the biochemical requirements of the organelle. In particular, one subunit of the Tic complex, Tic62, has ...

  16. A milk diet partly containing soy protein does not change growth but regulates jejunal proteins in young goats.

    Science.gov (United States)

    Kuhla, S; Rudolph, P E; Albrecht, D; Schoenhusen, U; Zitnan, R; Tomek, W; Huber, K; Voigt, J; Metges, C C

    2007-09-01

    Soy protein is known to alter intestinal function and structure. We determined in young goats whether a diet partly containing soy protein differently affects intestinal morphology and the jejunal and hepatic proteome as compared with a milk diet. Fourteen male 2-wk-old White German dairy goat kids were fed comparable diets based on whole cow's milk in which 35% of the crude protein was casein (milk protein group; MP) or soy protein supplemented by indispensable AA (SPAA) for 34 d (n = 7/group). Body weight gain and food efficiency were not different. Jejunal and hepatic tissue was collected to determine intestinal morphology by microscopy and protein repertoire by 2-dimensional gel electrophoresis and mass spectrometry. Jejunal crypt depth was reduced and villus height to crypt depth ratio was higher in SPAA than in milk protein. Out of 131 proteins identified, 32 proteins were found to be differently expressed in both groups. In SPAA, down-regulated jejunal proteins were involved in processes related to cytoskeleton generation, protein, lipid, and energy metabolism. Downregulated hepatic proteins were related to glycolysis and Krebs cycle. Thirteen proteins were upregulated in SPAA. Among these, 2 hepatic proteins were related to carbohydrate breakdown. The other 11 jejunal proteins were involved in cytoskeleton assembly, proteolysis, and carbohydrate breakdown. In addition, glutathione-S-transferase was found to be upregulated in the medial jejunum. In conclusion, a SPAA diet as compared with a milk diet was related to changes in jejunal morphology and jejunal proteins relevant for protein turnover, energy metabolism, and cytoskeleton assembly with no apparent impact on animal BW gain.

  17. Structural Elements Regulating AAA+ Protein Quality Control Machines

    Directory of Open Access Journals (Sweden)

    Chiung-Wen Chang

    2017-05-01

    Full Text Available Members of the ATPases Associated with various cellular Activities (AAA+ superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS motif, and the Pre-Sensor I insert (PS-I motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

  18. Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

    DEFF Research Database (Denmark)

    Mannervik, M; Fan, S; Ström, A C

    1999-01-01

    Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression...... of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation...... of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression...

  19. Regulated protein turnover: snapshots of the proteasome in action.

    Science.gov (United States)

    Bhattacharyya, Sucharita; Yu, Houqing; Mim, Carsten; Matouschek, Andreas

    2014-02-01

    The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.

  20. Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression.

    Science.gov (United States)

    David, Diana; Surendran, Arun; Thulaseedharan, Jissa V; Nair, Asha S

    2018-03-13

    Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation. CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines. Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf

  1. Inactivation, stabilization and redox regulation of iron-containing proteins

    NARCIS (Netherlands)

    Spee, J.H.

    1997-01-01

    Summary

    Microperoxidases: kinetics and stability.

    Microperoxidases are small enzymes prepared by proteolytic digestion of cytochromes c. The proteolytic removal of most of the protein environment allows these enzymes to use a

  2. Role of adaptor proteins in motor regulation and membrane transport

    NARCIS (Netherlands)

    M.A. Schlager (Max)

    2010-01-01

    markdownabstract__Abstract__ Active transport along the cytoskeleton is a process essential for proper cellular function. Although much is known about the motor proteins that generate the necessary force and the cytoskeleton that provides the cellular infrastructure, many questions still

  3. Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

    Science.gov (United States)

    Enjapoori, Ashwantha Kumar; Lefèvre, Christophe M; Nicholas, Kevin R; Sharp, Julie A

    2017-02-01

    Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins.

    Science.gov (United States)

    Gogate, Shilpa S; Nasser, Rena; Shapiro, Irving M; Risbud, Makarand V

    2011-07-01

    To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of β-1,3-glucuronyltransferase 1 (GlcAT-1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells. Real-time reverse transcriptase-polymerase chain reaction and Western blotting were used to measure GlcAT-1 expression. Transfections were performed to determine the effect of HIF-1α and HIF-2α on GlcAT-1 promoter activity. Under hypoxic conditions there was an increase in GlcAT-1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT-1 expression. Suppression of HIF-1α and HIF-2α induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1α as well as with CA-HIF-2α suppressed GlcAT-1 promoter activity only in nucleus pulposus cells, suggesting a cell type-specific regulation. Site-directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT-1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia-responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF-dependent repressor. Finally, we showed that hypoxic expression of GlcAT-1 was also partially dependent on MAPK signaling. These studies demonstrate that hypoxia regulates GlcAT-1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells. Copyright © 2011 by the American College of Rheumatology.

  5. The Alzheimer Amyloid Precursor Protein (APP) and Fe65, an APP-Binding Protein, Regulate Cell Movement

    Science.gov (United States)

    Sabo, Shasta L.; Ikin, Annat F.; Buxbaum, Joseph D.; Greengard, Paul

    2001-01-01

    FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with β1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound–healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility. PMID:11425871

  6. The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

    Science.gov (United States)

    Sabo, S L; Ikin, A F; Buxbaum, J D; Greengard, P

    2001-06-25

    FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.

  7. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity

    NARCIS (Netherlands)

    Kevenaar, Josta T|info:eu-repo/dai/nl/338771042; Bianchi, Sarah; van Spronsen, Myrrhe|info:eu-repo/dai/nl/337616655; Olieric, Natacha; Lipka, Joanna|info:eu-repo/dai/nl/369403142; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin|info:eu-repo/dai/nl/304075655; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C|info:eu-repo/dai/nl/298806630; de Graaff, Esther|info:eu-repo/dai/nl/148374646; Akhmanova, Anna|info:eu-repo/dai/nl/156410591; Steinmetz, Michel O; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502

    2016-01-01

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human

  8. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

    Science.gov (United States)

    Church, George M.; Esvelt, Kevin; Mali, Prashant

    2017-03-07

    Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

  9. CGGBP1 is a nuclear and midbody protein regulating abscission

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Umashankar, E-mail: umashankar.singh@genpat.uu.se; Westermark, Bengt

    2011-01-15

    Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.

  10. CGGBP1 is a nuclear and midbody protein regulating abscission

    International Nuclear Information System (INIS)

    Singh, Umashankar; Westermark, Bengt

    2011-01-01

    Abscission marks the completion of cell division and its failure is associated with delayed cytokinesis and even tetraploidization. Aberrant abscission and consequential ploidy changes can underlie various diseases including cancer. Midbody, a transient structure formed in the intercellular bridge during telophase, contains several proteins including Aurora kinase B (AURKB), which participate in abscission. We report here an unexpected expression pattern and function of the transcription repressor protein CGG triplet repeat-binding protein 1 (CGGBP1), in normal human fibroblasts. We show that CGGBP1, a chromatin-associated protein, trans-localizes to spindle midzone and midbodies in a manner similar to that of AURKB. CGGBP1 depletion resulted in a cell cycle block at G2, characterized by failure of cells to undergo mitosis and also reduced entry into S phase. Consistent with its presence in the midbodies, live microscopy showed that CGGBP1 deficiency caused mitotic failure at abscission resulting in tetraploidy, which could be rescued by CGGBP1 overexpression. These results show that CGGBP1 is a bona fide midbody protein required for normal abscission and mitosis in general.

  11. Supervised learning classification models for prediction of plant virus encoded RNA silencing suppressors.

    Directory of Open Access Journals (Sweden)

    Zeenia Jagga

    Full Text Available Viral encoded RNA silencing suppressor proteins interfere with the host RNA silencing machinery, facilitating viral infection by evading host immunity. In plant hosts, the viral proteins have several basic science implications and biotechnology applications. However in silico identification of these proteins is limited by their high sequence diversity. In this study we developed supervised learning based classification models for plant viral RNA silencing suppressor proteins in plant viruses. We developed four classifiers based on supervised learning algorithms: J48, Random Forest, LibSVM and Naïve Bayes algorithms, with enriched model learning by correlation based feature selection. Structural and physicochemical features calculated for experimentally verified primary protein sequences were used to train the classifiers. The training features include amino acid composition; auto correlation coefficients; composition, transition, and distribution of various physicochemical properties; and pseudo amino acid composition. Performance analysis of predictive models based on 10 fold cross-validation and independent data testing revealed that the Random Forest based model was the best and achieved 86.11% overall accuracy and 86.22% balanced accuracy with a remarkably high area under the Receivers Operating Characteristic curve of 0.95 to predict viral RNA silencing suppressor proteins. The prediction models for plant viral RNA silencing suppressors can potentially aid identification of novel viral RNA silencing suppressors, which will provide valuable insights into the mechanism of RNA silencing and could be further explored as potential targets for designing novel antiviral therapeutics. Also, the key subset of identified optimal features may help in determining compositional patterns in the viral proteins which are important determinants for RNA silencing suppressor activities. The best prediction model developed in the study is available as a

  12. Coincident light and clock regulation of pseudoresponse regulator protein 37 (PRR37) controls photoperiodic flowering in sorghum

    Science.gov (United States)

    Variation in flowering time was essential during widespread crop domestication and optimal timing of reproduction remains critical to modern agriculture. Ma1, the major repressor of flowering in sorghum in long days, was identified as the pseudo-response regulator protein PRR37. Three prr37 allele...

  13. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins

  14. Indispensable roles of mammalian Cbl family proteins as negative regulators of protein tyrosine kinase signaling: Insights from in vivo models

    OpenAIRE

    Naramura, Mayumi; Band, Vimla; Band, Hamid

    2011-01-01

    All higher eukaryotes utilize protein tyrosine kinases (PTKs) as molecular switches to control a variety of cellular signals. Notably, many PTKs have been identified as proto-oncogenes whose aberrant expression, mutations or co-option by pathogens can lead to human malignancies. Thus, it is obvious that PTK functions must be precisely regulated in order to maintain homeostasis of an organism. Investigations over the past fifteen years have revealed that members of the Cbl family proteins can ...

  15. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

  16. Functional Characterization of the Canine Heme-Regulated eIF2α Kinase: Regulation of Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Kimon C. Kanelakis

    2009-01-01

    Full Text Available The heme-regulated inhibitor (HRI negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α (eIF2α thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

  17. Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

    OpenAIRE

    E. Ruchti; P.J. Roach; A.A. DePaoli-Roach; P.J. Magistretti; I. Allaman

    2016-01-01

    The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the ...

  18. Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

    OpenAIRE

    Chen, Qingfeng; Chen, Yi-Ping Phoebe

    2006-01-01

    Abstract Background AMP-activated protein kinase (AMPK) has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data ar...

  19. The tumor suppressors p33ING1 and p33ING2 interact with alien in vivo and enhance alien-mediated gene silencing.

    Science.gov (United States)

    Fegers, Inga; Kob, Robert; Eckey, Maren; Schmidt, Oliver; Goeman, Frauke; Papaioannou, Maria; Escher, Niko; von Eggeling, Ferdinand; Melle, Christian; Baniahmad, Aria

    2007-11-01

    The tumor suppressor p33ING1 is involved in DNA repair and cell cycle regulation. Furthermore, p33ING1 is a transcriptional silencer that recognizes the histone mark for trimethylated lysine 4 at histone H3. Interestingly, expression of p33ING1 and p33ING2 is able to induce premature senescence in primary human fibroblasts. The corepressor Alien is involved in gene silencing mediated by selected members of nuclear hormone receptors. In addition, Alien acts as a corepressor for E2F1, a member of the E2F cell cycle regulatory family. Furthermore, recent findings suggest that Alien is complexed with transcription factors participating in DNA repair and chromatin. Here, using a proteomic approach by surface-enhanced laser desorption ionization and mass spectrometry (SELDI-MS) combined with immunological techniques, we show that Alien interacts in vivo with the tumor suppressor p33ING1 as well as with the related tumor suppressor candidate p33ING2. The interaction of Alien with p33ING1 and p33ING2 was confirmed in vitro with GST-pull-down, suggesting a direct binding of Alien to these factors. The binding domain was mapped to a central region of Alien. Functionally, the expression of p33ING1 or p33ING2 enhances the Alien-mediated silencing, suggesting that the interaction plays a role in transcriptional regulation. Thus, the findings suggest that the identified interaction between Alien and the tumor suppressors p33ING1 and p33ING2 reveals a novel cellular protein network.

  20. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    detoxification. Plant Noduline 26-like Intrinsic Proteins (NIPs) can channel As(III) and consequently influence the detoxification process. The role of the Tonoplast Intrinsic Proteins (TIPs) in As(III) detoxification remains to be clarified, yet TIPs could have an impact on As(III) accumulation in plant cell...... to development of plants with levated arsenic tolerance. Plants with the ability to hyperaccumulate arsenic could find its use in soil remediation while crop plants with efficient arsenic detoxification mechanisms could be used for food production in areas polluted with arsenic....

  1. A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu–Gli protein complexes

    OpenAIRE

    Tukachinsky, Hanna; Lopez, Lyle V.; Salic, Adrian

    2010-01-01

    In vertebrates, Hedgehog (Hh) signaling initiated in primary cilia activates the membrane protein Smoothened (Smo) and leads to activation of Gli proteins, the transcriptional effectors of the pathway. In the absence of signaling, Gli proteins are inhibited by the cytoplasmic protein Suppressor of Fused (SuFu). It is unclear how Hh activates Gli and whether it directly regulates SuFu. We find that Hh stimulation quickly recruits endogenous SuFu–Gli complexes to cilia, suggesting a model in wh...

  2. V2 from a curtovirus is a suppressor of post-transcriptional gene silencing.

    Science.gov (United States)

    Luna, Ana P; Rodríguez-Negrete, Edgar A; Morilla, Gabriel; Wang, Liping; Lozano-Durán, Rosa; Castillo, Araceli G; Bejarano, Eduardo R

    2017-10-01

    The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway.

  3. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator o...

  4. Focus on PTEN regulation

    Directory of Open Access Journals (Sweden)

    Miriam eBermudez-Brito

    2015-07-01

    Full Text Available The role of PTEN as a tumour suppressor has been for a long time attributed to its lipid phosphatase activity against PI(3,4,5P3, the phospholipid product of the class I PI3Ks. Besides its traditional role as a lipid phosphatase at the plasma membrane, a wealth of data has shown that PTEN can function independently of its phosphatase activity and that PTEN also exists and plays a role in the nucleus, in cytoplasmic organelles and extracellularly. Accumulating evidence has shed light on diverse physiological functions of PTEN which are accompanied by a complex regulation of its expression and activity. PTEN levels and function are regulated transcriptionally, post-transcriptionally and post-translationally. PTEN is also sensitive to regulation by its interacting proteins and its localization. Herein, we summarize the current knowledge on mechanisms that regulate the expression and enzymatic activity of PTEN and its role in human diseases.

  5. Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

    Science.gov (United States)

    Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

    2018-02-15

    The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Genome-wide regulation of TATA-binding protein activity

    NARCIS (Netherlands)

    van Werven, F.J.

    2009-01-01

    Transcription, the synthesis of RNA from a DNA template, is a well-controlled process. TATA binding protein (TBP) recruitment to promoters is essential for transcription by all three RNA polymerases, and often is the rate-limiting step of transcription initiation. TBP is incorporated into different

  7. Regulation of Cellular and Molecular Functions by Protein ...

    Indian Academy of Sciences (India)

    phosphorylation of histidine, lysine and arginine is also known to occur .... A protein tyrosine kinase TK-l isolated from rat spleen phos- phorylates itself ... GENERAL I ARTICLE. Experiment. Unphosphorylated TK-l. Phosphorylated TK-l. Phosphorylated TK-I + phosphatase. Rephosphorylated TK-I. Kinase activity. 11. 100. 13.

  8. Regulation of triglyceride metabolism by angiopoietin-like proteins

    NARCIS (Netherlands)

    Mattijssen, F.B.J.; Kersten, A.H.

    2012-01-01

    asma triglyceride concentrations are determined by the balance between production of the triglyceride-rich lipoproteins VLDL and chylomicrons in liver and intestine, and their lipoprotein lipase-mediated clearance in peripheral tissues. In the last decade, the group of Angiopoietin-like proteins has

  9. Regulation of neuronal differentiation by proteins associated with nuclear bodies.

    Directory of Open Access Journals (Sweden)

    Benjamin Förthmann

    Full Text Available Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN protein localizes to Cajal bodies (CBs and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA. How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23 is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs. Here we demonstrate that FGF-2(23 blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23-dependent transcription. Our results indicate that FGF-2(23 and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs. In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs. The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.

  10. Nitrogen regulation of protein-protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea.

    Science.gov (United States)

    Pedro-Roig, Laia; Lange, Christian; Bonete, María José; Soppa, Jörg; Maupin-Furlow, Julie

    2013-10-01

    Gene homologs of GlnK PII regulators and AmtB-type ammonium transporters are often paired on prokaryotic genomes, suggesting these proteins share an ancient functional relationship. Here, we demonstrate for the first time in Archaea that GlnK associates with AmtB in membrane fractions after ammonium shock, thus, providing a further insight into GlnK-AmtB as an ancient nitrogen sensor pair. For this work, Haloferax mediterranei was advanced for study through the generation of a pyrE2-based counterselection system that was used for targeted gene deletion and expression of Flag-tagged proteins from their native promoters. AmtB1-Flag was detected in membrane fractions of cells grown on nitrate and was found to coimmunoprecipitate with GlnK after ammonium shock. Thus, in analogy to bacteria, the archaeal GlnK PII may block the AmtB1 ammonium transporter under nitrogen-rich conditions. In addition to this regulated protein-protein interaction, the archaeal amtB-glnK gene pairs were found to be highly regulated by nitrogen availability with transcript levels high under conditions of nitrogen limitation and low during nitrogen excess. While transcript levels of glnK-amtB are similarly regulated by nitrogen availability in bacteria, transcriptional regulators of the bacterial glnK promoter including activation by the two-component signal transduction proteins NtrC (GlnG, NRI) and NtrB (GlnL, NRII) and sigma factor σ(N) (σ(54) ) are not conserved in archaea suggesting a novel mechanism of transcriptional control. © 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  11. Regulation of activity of the yeast TATA-binding protein through intra ...

    Indian Academy of Sciences (India)

    Unknown

    413. Keywords. Chemical cross-linking; dimerization; gene regulation; TBP; transcription; yeast ... [Vanathi P, Mishra A K and Bhargava P 2003 Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions; J. Biosci. ..... Data given was obtained from more than three independent estimations in ...

  12. Identification of Hypoxia-Regulated Proteins Using MALDI-Mass Spectrometry Imaging Combined with Quantitative Proteomics

    DEFF Research Database (Denmark)

    Djidja, Marie-Claude; Chang, Joan; Hadjiprocopis, Andreas

    2014-01-01

    -regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC...... data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed....

  13. Long-Timescale Dynamics and Regulation of Sec-Facilitated Protein Translocation

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2012-10-01

    Full Text Available We present a coarse-grained modeling approach that spans the nanosecond- to minute-timescale dynamics of cotranslational protein translocation. The method enables direct simulation of both integral membrane protein topogenesis and transmembrane domain (TM stop-transfer efficiency. Simulations reveal multiple kinetic pathways for protein integration, including a mechanism in which the nascent protein undergoes slow-timescale reorientation, or flipping, in the confined environment of the translocon channel. Competition among these pathways gives rise to the experimentally observed dependence of protein topology on ribosomal translation rate and protein length. We further demonstrate that sigmoidal dependence of stop-transfer efficiency on TM hydrophobicity arises from local equilibration of the TM across the translocon lateral gate, and it is predicted that slowing ribosomal translation yields decreased stop-transfer efficiency in long proteins. This work reveals the balance between equilibrium and nonequilibrium processes in protein targeting, and it provides insight into the molecular regulation of the Sec translocon.

  14. Fat-specific protein 27 regulates storage of triacylglycerol

    DEFF Research Database (Denmark)

    Keller, P.; Petrie, J.T.; Rose, P. De

    2008-01-01

    FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins....... In this study, we investigated the function of another member of this family, FSP27 (Cidec), in apoptosis and adipocyte metabolism. Although overexpression of FSP27 is sufficient to increase apoptosis of 293T and 3T3-L1 cells, more physiological levels of expression stimulate spontaneous lipid accumulation...... in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L...

  15. The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS.

    Science.gov (United States)

    Ito, Toshiro; Wellmer, Frank; Yu, Hao; Das, Pradeep; Ito, Natsuko; Alves-Ferreira, Márcio; Riechmann, José Luis; Meyerowitz, Elliot M

    2004-07-15

    The Arabidopsis homeotic gene AGAMOUS (AG) is necessary for the specification of reproductive organs (stamens and carpels) during the early steps of flower development. AG encodes a transcription factor of the MADS-box family that is expressed in stamen and carpel primordia. At later stages of development, AG is expressed in distinct regions of the reproductive organs. This suggests that AG might function during the maturation of stamens and carpels, as well as in their early development. However, the developmental processes that AG might control during organogenesis and the genes that are regulated by this factor are largely unknown. Here we show that microsporogenesis, the process leading to pollen formation, is induced by AG through activation of the SPOROCYTELESS gene (SPL, also known as NOZZLE,NZZ), a regulator of sporogenesis. Furthermore, we demonstrate that SPL can induce microsporogenesis in the absence of AG function, suggesting that AG controls a specific process during organogenesis by activating another regulator that performs a subset of its functions.

  16. Structural aspects of protein kinase ASK1 regulation

    Czech Academy of Sciences Publication Activity Database

    Obšil, Tomáš; Obšilová, Veronika

    2017-01-01

    Roč. 66, 1 Dec (2017), s. 31-36 ISSN 2212-4926 R&D Projects: GA ČR(CZ) GA16-02739S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ASK1 kinase * apoptosis * thioredoxin * 14-3-3 protein Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  17. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  18. Tumor Suppressors and Breast Cancer

    National Research Council Canada - National Science Library

    Shyam, E

    2002-01-01

    .... Both BRCA2 and BRCA1 proteins were shown to have transcriptional activation domains and also shown to be associated with RNA polymerase suggesting that these proteins may function as transcriptional...

  19. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Science.gov (United States)

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.

    Science.gov (United States)

    Iurlaro, Mario; Ficz, Gabriella; Oxley, David; Raiber, Eun-Ang; Bachman, Martin; Booth, Michael J; Andrews, Simon; Balasubramanian, Shankar; Reik, Wolf

    2013-01-01

    DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts. We compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex). Our screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.

  1. BET bromodomain proteins and epigenetic regulation of inflammation: implications for type 2 diabetes and breast cancer.

    Science.gov (United States)

    Nicholas, Dequina A; Andrieu, Guillaume; Strissel, Katherine J; Nikolajczyk, Barbara S; Denis, Gerald V

    2017-01-01

    Chronic inflammation drives pathologies associated with type 2 diabetes (T2D) and breast cancer. Obesity-driven inflammation may explain increased risk and mortality of breast cancer with T2D reported in the epidemiology literature. Therapeutic approaches to target inflammation in both T2D and cancer have so far fallen short of the expected improvements in disease pathogenesis or outcomes. The targeting of epigenetic regulators of cytokine transcription and cytokine signaling offers one promising, untapped approach to treating diseases driven by inflammation. Recent work has deeply implicated the Bromodomain and Extra-Terminal domain (BET) proteins, which are acetylated histone "readers", in epigenetic regulation of inflammation. This review focuses on inflammation associated with T2D and breast cancer, and the possibility of targeting BET proteins as an approach to regulating inflammation in the clinic. Understanding inflammation in the context of BET protein regulation may provide a basis for designing promising therapeutics for T2D and breast cancer.

  2. Novel function of the retinoblastoma protein in fat: regulation of white versus brown adipocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Jacob B; te Riele, Hein; Kristiansen, Karsten

    2004-01-01

    the major energy store and brown adipocytes being potent energy-dissipaters through thermogenesis. Yet, little is known about factors differentially regulating the formation of white and brown fat cells. Members of the retinoblastoma protein family (pRB, p107, p130) have been implicated in the regulation...... of adipocyte differentiation, and expression and phosphorylation of the three retinoblastoma family proteins oscillate in a characteristic manner during differentiation of the white preadipocyte cell line 3T3-L1. We have recently demonstrated a surprising function of the retinoblastoma protein...... in the regulation of white versus brown adipocyte differentiation in vitro and possibly in vivo. Here we summarize the current knowledge on the retinoblastoma protein in fat cells, with particular emphasis on its potential role in adipocyte lineage commitment and differentiation....

  3. Hormone response element binding proteins: novel regulators of vitamin D and estrogen signaling.

    Science.gov (United States)

    Lisse, Thomas S; Hewison, Martin; Adams, John S

    2011-03-01

    Insights from vitamin D-resistant New World primates and their human homologues as models of natural and pathological insensitivity to sterol/steroid action have uncovered a family of novel intracellular vitamin D and estrogen regulatory proteins involved in hormone action. The proteins, known as "vitamin D or estrogen response element-binding proteins", behave as potent cis-acting, transdominant regulators to inhibit steroid receptor binding to DNA response elements and is responsible for vitamin D and estrogen resistances. This set of interactors belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family of previously known pre-mRNA-interacting proteins. This review provides new insights into the mechanism by which these novel regulators of signaling and metabolism can act to regulate responses to vitamin D and estrogen. In addition the review also describes other molecules that are known to influence nuclear receptor signaling through interaction with hormone response elements. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. The Role of BCL2 Family of Apoptosis Regulator Proteins in Acute and Chronic Leukemias

    Directory of Open Access Journals (Sweden)

    Flora Tzifi

    2012-01-01

    Full Text Available The disturbance of apoptosis molecular signaling pathways is involved in carcinogenesis. BCL2 family of proteins is the hallmark of apoptosis regulation. In the last decade, new members of BCL2 gene family were discovered and cloned and were found to be differentially expressed in many types of cancer. BCL2 protein family, through its role in regulation of apoptotic pathways, is possibly related to cancer pathophysiology and resistance to conventional chemotherapy. It is well known that leukemias are haematopoietic malignancies characterized by biological diversity, varied cytogenetics, different immunophenotype profiles, and diverse outcome. Current research focuses on the prognostic impact and specific role of these proteins in the pathogenesis of leukemias. The understanding of the molecular pathways that participate in the biology of leukemias may lead to the design of new therapies which may improve patients' survival. In the present paper, we describe current knowledge on the role of BCL2 apoptosis regulator proteins in acute and chronic leukemias.

  5. Identification and network of outer membrane proteins regulating streptomysin resistance in Escherichia coli.

    Science.gov (United States)

    Li, Hui; Wang, Bao-Cheng; Xu, Wen-Jiao; Lin, Xiang-Min; Peng, Xuan-Xian

    2008-09-01

    Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, Delta tolC, Delta ompT, Delta dps, Delta tolC-R, Delta ompT-R, Delta dps-R and Delta fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli.

  6. The PTEN tumor suppressor gene and its role in lymphoma pathogenesis

    Science.gov (United States)

    Wang, Xiaoxiao; Huang, Huiqiang; Young, Ken H.

    2015-01-01

    The phosphatase and tensin homolog gene PTEN is one of the most frequently mutated tumor suppressor genes in human cancer. Loss of PTEN function occurs in a variety of human cancers via its mutation, deletion, transcriptional silencing, or protein instability. PTEN deficiency in cancer has been associated with advanced disease, chemotherapy resistance, and poor survival. Impaired PTEN function, which antagonizes phosphoinositide 3-kinase (PI3K) signaling, causes the accumulation of phosphatidylinositol (3,4,5)-triphosphate and thereby the suppression of downstream components of the PI3K pathway, including the protein kinase B and mammalian target of rapamycin kinases. In addition to having lipid phosphorylation activity, PTEN has critical roles in the regulation of genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration. Although PTEN deficiency in solid tumors has been studied extensively, rare studies have investigated PTEN alteration in lymphoid malignancies. However, genomic or epigenomic aberrations of PTEN and dysregulated signaling are likely critical in lymphoma pathogenesis and progression. This review provides updated summary on the role of PTEN deficiency in human cancers, specifically in lymphoid malignancies; the molecular mechanisms of PTEN regulation; and the distinct functions of nuclear PTEN. Therapeutic strategies for rescuing PTEN deficiency in human cancers are proposed. PMID:26655726

  7. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  8. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    Arsenic is a metalloid that is toxic to living organisms. The use of arsenic-contaminated ground water for drinking and for irrigation in agriculture presents serious health problems for millions of people in many parts of the world. Arsenate (As(V)) and arsenite (As(III)), the two most widespread...... in Arabidopsis. The function of N-terminus in regulation of AtNIP5;1 in planta remains elusive. ICPMS analysis of the elemental composition and expression analysis did not clarify the role of Nterminus of AtNIP5;1 in arsenic accumulation in Arabidopsis. Further research is needed to elucidate the role of N...

  10. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  11. A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C elegans.

    Science.gov (United States)

    Srinivasan, Dayalan G; Fisk, Ridgely M; Xu, Huihong; van den Heuvel, Sander

    2003-05-15

    The Caenorhabditis elegans coiled-coil protein LIN-5 mediates several processes in cell division that depend on spindle forces, including alignment and segregation of chromosomes and positioning of the spindle. Here, we describe two closely related proteins, GPR-1 and GPR-2 (G protein regulator), which associate with LIN-5 in vivo and in vitro and depend on LIN-5 for localization to the spindle and cell cortex. GPR-1/GPR-2 contain a GoLoco/GPR motif that mediates interaction with GDP-bound Galpha(i/o). Inactivation of lin-5, gpr-1/gpr-2, or the Galpha(i/o) genes goa-1 and gpa-16 all cause highly similar chromosome segregation and spindle positioning defects, indicating a positive role for the LIN-5 and GPR proteins in G protein signaling. The lin-5 and gpr-1/gpr-2 genes appear to act downstream of the par polarity genes in the one- and two-cell stages and downstream of the tyrosine kinase-related genes mes-1 and src-1 at the four-cell stage. Together, these results indicate that GPR-1/GPR-2 in association with LIN-5 activate G protein signaling to affect spindle force. Polarity determinants may regulate LIN-5/GPR/Galpha locally to create the asymmetric forces that drive spindle movement. Results in C. elegans and other species are consistent with a novel model for receptor-independent activation of Galpha(i/o) signaling.

  12. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

    International Nuclear Information System (INIS)

    Born, E J; Hartman, S V; Holstein, S A

    2013-01-01

    Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

  14. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk

    2017-10-01

    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  15. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... on protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  16. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    Science.gov (United States)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  17. Tumor suppressor QM-like gene from disk abalone (Haliotis discus discus): molecular characterization and transcriptional analysis upon immune challenge.

    Science.gov (United States)

    Oh, Chulhong; De Zoysa, Mahanama; Nikapitiya, Chamilani; Whang, Ilson; Kim, Yu Cheol; Kang, Do-Hyung; Heo, Soo-jin; Choi, Young-Ung; Choi, Cheol Young; Lee, Jae-Seong; Lee, Jehee

    2010-09-01

    We describe molecular characterization and transcriptional analysis of the gene encoding tumor suppressor QM-like protein, AbQM, in the disk abalone Haliotis discus discus. The full-length cDNA (765-bp) of AbQM was found to consist of a 654-bp ORF coding for a 218 amino acid protein of a 25 kDa molecular mass with a 10.2 isoelectric point. Analysis of AbQM sequence revealed the presence of characteristic motifs, including the ribosomal protein L10 signature, SH3-binding motif and two antibiotic binding sites. Phylogenetic analysis confirmed that AbQM is closely related to other mollusk QM proteins, and altogether they form a mollusk QM protein sub-family which displays evolutionary conservation from yeast to human. Tissue-specific expression and transcriptional regulation of AbQM was analyzed by quantitative real-time PCR in response to bacterial (Vibrio alginolyticus and Vibrio parahemolyticus, Listeria monocytogenes) and viral (viral hemorrhagic septicemia virus, VHSV) challenge. AbQM transcripts were found to be expressed ubiquitously in all examined tissues in a constitutive manner, as similar expression levels were detected in hemocytes, mantle, digestive tract and muscle. Upon bacterial and VHSV challenge, AbQM showed significant up-regulation in gills, but not in hemocytes. Taken together, these findings suggest that AbQM in abalone-like mollusks can respond to and facilitate a defensive effect against pathogenic infection. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Distinct and competitive regulatory patterns of tumor suppressor genes and oncogenes in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Min Zhao

    Full Text Available So far, investigators have found numerous tumor suppressor genes (TSGs and oncogenes (OCGs that control cell proliferation and apoptosis during cancer development. Furthermore, TSGs and OCGs may act as modulators of transcription factors (TFs to influence gene regulation. A comprehensive investigation of TSGs, OCGs, TFs, and their joint target genes at the network level may provide a deeper understanding of the post-translational modulation of TSGs and OCGs to TF gene regulation.In this study, we developed a novel computational framework for identifying target genes of TSGs and OCGs using TFs as bridges through the integration of protein-protein interactions and gene expression data. We applied this pipeline to ovarian cancer and constructed a three-layer regulatory network. In the network, the top layer was comprised of modulators (TSGs and OCGs, the middle layer included TFs, and the bottom layer contained target genes. Based on regulatory relationships in the network, we compiled TSG and OCG profiles and performed clustering analyses. Interestingly, we found TSGs and OCGs formed two distinct branches. The genes in the TSG branch were significantly enriched in DNA damage and repair, regulating macromolecule metabolism, cell cycle and apoptosis, while the genes in the OCG branch were significantly enriched in the ErbB signaling pathway. Remarkably, their specific targets showed a reversed functional enrichment in terms of apoptosis and the ErbB signaling pathway: the target genes regulated by OCGs only were enriched in anti-apoptosis and the target genes regulated by TSGs only were enriched in the ErbB signaling pathway.This study provides the first comprehensive investigation of the interplay of TSGs and OCGs in a regulatory network modulated by TFs. Our application in ovarian cancer revealed distinct regulatory patterns of TSGs and OCGs, suggesting a competitive regulatory mechanism acting upon apoptosis and the ErbB signaling pathway through

  19. Acid-regulated proteins induced by Streptococcus mutans and other oral bacteria during acid shock.

    Science.gov (United States)

    Hamilton, I R; Svensäter, G

    1998-10-01

    Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the

  20. Regulation of NADPH oxidase 5 by protein kinase C isoforms.

    Directory of Open Access Journals (Sweden)

    Feng Chen

    Full Text Available NADPH oxidase5 (Nox5 is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular

  1. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  2. AZU-1: A Candidate Breast Tumor Suppressor and Biomarker for Tumor Progression

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Huei-Mei; Schmeichel, Karen L; Mian, I. Saira; Lelie`vre, Sophie; Petersen, Ole W; Bissell, Mina J

    2000-02-04

    To identify genes misregulated in the final stages of breast carcinogenesis, we performed differential display to compare the gene expression patterns of the human tumorigenic mammary epithelial cells, HMT-3522-T4-2, with those of their immediate premalignant progenitors, HMT-3522-S2. We identified a novel gene, called anti-zuai-1 (AZU-1), that was abundantly expressed in non- and premalignant cells and tissues but was appreciably reduced in breast tumor cell types and in primary tumors. The AZU-1 gene encodes an acidic 571-amino-acid protein containing at least two structurally distinct domains with potential protein-binding functions: an N-terminal serine and proline-rich domain with a predicted immunoglobulin-like fold and a C-terminal coiled-coil domain. In HMT-3522 cells, the bulk of AZU-1 protein resided in a detergent-extractable cytoplasmic pool and was present at much lower levels in tumorigenic T4-2 cells than in their nonmalignant counterparts. Reversion of the tumorigenic phenotype of T4-2 cells, by means described previously, was accompanied by the up-regulation of AZU-1. In addition, reexpression of AZU-1 in T4-2 cells, using viral vectors, was sufficient to reduce their malignant phenotype substantially, both in culture and in vivo. These results indicate that AZU-1 is a candidate breast tumor suppressor that may exert its effects by promoting correct tissue morphogenesis.

  3. LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

    Science.gov (United States)

    Cai, Ting-Ting; Ye, Shu-Biao; Liu, Yi-Na; He, Jia; Chen, Qiu-Yan; Mai, Hai-Qiang; Zhang, Chuan-Xia; Cui, Jun; Zhang, Xiao-Shi; Zeng, Yi-Xin

    2017-01-01

    Myeloid-derived suppressor cells (MDSCs) are expanded in tumor microenvironments, including that of Epstein–Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). The link between MDSC expansion and EBV infection in NPC is unclear. Here, we show that EBV latent membrane protein 1 (LMP1) promotes MDSC expansion in the tumor microenvironment by promoting extra-mitochondrial glycolysis in malignant cells, which is a scenario for immune escape initially suggested by the frequent, concomitant detection of abundant LMP1, glucose transporter 1 (GLUT1) and CD33+ MDSCs in tumor sections. The full process has been reconstituted in vitro. LMP1 promotes the expression of multiple glycolytic genes, including GLUT1. This metabolic reprogramming results in increased expression of the Nod-like receptor family protein 3 (NLRP3) inflammasome, COX-2 and P-p65 and, consequently, increased production of IL-1β, IL-6 and GM-CSF. Finally, these changes in the environment of malignant cells result in enhanced NPC-derived MDSC induction. One key step is the physical interaction of LMP1 with GLUT1 to stabilize the GLUT1 protein by blocking its K48-ubiquitination and p62-dependent autolysosomal degradation. This work indicates that LMP1-mediated glycolysis regulates IL-1β, IL-6 and GM-CSF production through the NLRP3 inflammasome, COX-2 and P-p65 signaling pathways to enhance tumor-associated MDSC expansion, which leads to tumor immunosuppression in NPC. PMID:28732079

  4. TIS11 Family Proteins and Their Roles in Posttranscriptional Gene Regulation

    Directory of Open Access Journals (Sweden)

    Maria Baou

    2009-01-01

    Full Text Available Posttranscriptional regulation of gene expression of mRNAs containing adenine-uridine rich elements (AREs in their 3 untranslated regions is mediated by a number of different proteins that interact with these elements to either stabilise or destabilise them. The present review concerns the TPA-inducible sequence 11 (TIS11 protein family, a small family of proteins, that appears to interact with ARE-containing mRNAs and promote their degradation. This family of proteins has been extensively studied in the past decade. Studies have focussed on determining their biochemical functions, identifying their target mRNAs, and determining their roles in cell functions and diseases.