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Sample records for suppressor protein caveolin-1

  1. Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells.

    PubMed

    Bourseau-Guilmain, E; Menard, J A; Lindqvist, E; Indira Chandran, V; Christianson, H C; Cerezo Magaña, M; Lidfeldt, J; Marko-Varga, G; Welinder, C; Belting, M

    2016-01-01

    Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment. PMID:27094744

  2. Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells

    PubMed Central

    Bourseau-Guilmain, E.; Menard, J. A.; Lindqvist, E.; Indira Chandran, V.; Christianson, H. C.; Cerezo Magaña, M.; Lidfeldt, J.; Marko-Varga, G.; Welinder, C.; Belting, M.

    2016-01-01

    Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment. PMID:27094744

  3. Calcium regulates caveolin-1 expression at the transcriptional level

    SciTech Connect

    Yang, Xiao-Yan; Huang, Cheng-Cheng; Kan, Qi-Ming; Li, Yan; Liu, Dan; Zhang, Xue-Cheng; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Caveolin-1 expression is regulated by calcium signaling at the transcriptional level. Black-Right-Pointing-Pointer An inhibitor of or siRNA to L-type calcium channel suppressed caveolin-1 expression. Black-Right-Pointing-Pointer Cyclosporine A or an NFAT inhibitor markedly reduced caveolin-1 expression. Black-Right-Pointing-Pointer Caveolin-1 regulation by calcium signaling is observed in several mouse cell lines. -- Abstract: Caveolin-1, an indispensable component of caveolae serving as a transformation suppressor protein, is highly expressed in poorly metastatic mouse osteosarcoma FBJ-S1 cells while highly metastatic FBJ-LL cells express low levels of caveolin-1. Calcium concentration is higher in FBJ-S1 cells than in FBJ-LL cells; therefore, we investigated the possibility that calcium signaling positively regulates caveolin-1 in mouse FBJ-S1 cells. When cells were treated with the calcium channel blocker nifedipine, cyclosporin A (a calcineurin inhibitor), or INCA-6 (a nuclear factor of activated T-cells [NFAT] inhibitor), caveolin-1 expression at the mRNA and protein levels decreased. RNA silencing of voltage-dependent L-type calcium channel subunit alpha-1C resulted in suppression of caveolin-1 expression. This novel caveolin-1 regulation pathway was also identified in mouse NIH 3T3 cells and Lewis lung carcinoma cells. These results indicate that caveolin-1 is positively regulated at the transcriptional level through a novel calcium signaling pathway mediated by L-type calcium channel/Ca{sup 2+}/calcineurin/NFAT.

  4. Identification of a novel domain at the N terminus of caveolin-1 that controls rear polarization of the protein and caveolae formation.

    PubMed

    Sun, Xing-Hui; Flynn, Daniel C; Castranova, Vincent; Millecchia, Lyndell L; Beardsley, Andrew R; Liu, Jun

    2007-03-01

    When cells are migrating, caveolin-1, the principal protein component of caveolae, is excluded from the leading edge and polarized at the cell rear. The dynamic feature depends on a specific sequence motif that directs intracellular trafficking of the protein. Deletion mutation analysis revealed a putative polarization domain at the N terminus of caveolin-1, between amino acids 32-60. Alanine substitution identified a minimal sequence of 10 residues ((46)TKEIDLVNRD(55)) necessary for caveolin-1 rear polarization. Interestingly, deletion of amino acids 1-60 did not prevent the polarization of caveolin-1 in human umbilical vein endothelial cells or wild-type mouse embryonic fibroblasts because of an interaction of Cav(61-178) mutant with endogenous caveolin-1. Surprisingly, expression of the depolarization mutant in caveolin-1 null cells dramatically impeded caveolae formation. Furthermore, knockdown of caveolae formation by methyl-beta-cyclodextrin failed to prevent wild-type caveolin-1 rear polarization. Importantly, genetic depletion of caveolin-1 led to disoriented migration, which can be rescued by full-length caveolin-1 but not the depolarization mutant, indicating a role of caveolin-1 polarity in chemotaxis. Thus, we have identified a sequence motif that is essential for caveolin-1 rear polarization and caveolae formation. PMID:17213184

  5. Regulation of Cellular Caveolin-1 Protein Expression in Murine Macrophages by Microbial Products

    PubMed Central

    Lei, Mei G.; Tan, Xiaoyu; Qureshi, Nilofer; Morrison, David C.

    2005-01-01

    Previously, we reported that expression of caveolin-1 in elicited peritoneal mouse macrophages was up-regulated by remarkably low (1.0-pg/ml) concentrations of Escherichia coli O111 lipopolysaccharide (LPS). Here we report that increases in caveolin-1 expression are manifested by different types of LPS, LPS-mimetic taxol, and heat-killed E. coli and to a much lesser extent by zymosan, polysaccharide-peptidoglycan, and heat-killed Staphylococcus aureus. Rhodobacter sphaeroides lipid A (RsDPLA) could not induce caveolin-1 expression in macrophages. Interestingly, polymyxin B (5 μg/ml) and RsDPLA show only a limited capacity to inhibit LPS-induced caveolin-1 expression. These findings suggest that expression of caveolin-1 in response to LPS may only partially be dependent upon lipid A. Recombinant tumor necrosis factor alpha marginally induces caveolin-1, suggesting that the ability of LPS to regulate caveolin-1 is not mediated primarily through an autocrine/paracrine mechanism involving this cytokine. Under conditions in which cellular levels of caveolin-1 are profoundly induced, no significant changes in TLR4 expression are observed. Of interest, caveolin-1 appears to localize to two cellular compartments, one associated with lipid rafts and a second associated with TLR4. Gamma interferon treatment inhibits the induction of caveolin-1 by LPS in macrophages. Inhibition of the p38 kinase-dependent pathway, but not the extracellular signal-regulated kinase pathway, effectively reduced the ability of LPS to mediate caveolin-1 up-regulation. Lactacystin, a potent inhibitor of the proteasome pathway, significantly modulates LPS-independent caveolin-1 expression, and lactacystin inhibits LPS-triggered caveolin-1 responses. These studies suggest that caveolin-1 up-regulation in response to LPS is likely to be proteasome dependent and triggered through the p38 kinase pathway. PMID:16299308

  6. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    SciTech Connect

    Ohnuma, Kei; Uchiyama, Masahiko; Hatano, Ryo; Takasawa, Wataru; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  7. The caveolin-1 connection to cell death and survival.

    PubMed

    Quest, A F G; Lobos-González, L; Nuñez, S; Sanhueza, C; Fernández, J-G; Aguirre, A; Rodríguez, D; Leyton, L; Torres, V

    2013-02-01

    Caveolins are a family of membrane proteins required for the formation of small plasma membrane invaginations called caveolae that are implicated in cellular trafficking processes. In addition to this structural role, these scaffolding proteins modulate numerous intracellular signaling pathways; often via direct interaction with specific binding partners. Caveolin-1 is particularly well-studied in this respect and has been attributed a large variety of functions. Thus, Caveolin-1 also represents the best-characterized isoform of this family with respect to its participation in cancer. Rather strikingly, available evidence indicates that Caveolin-1 belongs to a select group of proteins that function, depending on the cellular settings, both as tumor suppressor and promoter of cellular traits commonly associated with enhanced malignant behavior, such as metastasis and multi-drug resistance. The mechanisms underlying such ambiguity in Caveolin-1 function constitute an area of great interest. Here, we will focus on discussing how Caveolin-1 modulates cell death and survival pathways and how this may contribute to a better understanding of the ambiguous role this protein plays in cancer.

  8. The caveolin-1 connection to cell death and survival.

    PubMed

    Quest, A F G; Lobos-González, L; Nuñez, S; Sanhueza, C; Fernández, J-G; Aguirre, A; Rodríguez, D; Leyton, L; Torres, V

    2013-02-01

    Caveolins are a family of membrane proteins required for the formation of small plasma membrane invaginations called caveolae that are implicated in cellular trafficking processes. In addition to this structural role, these scaffolding proteins modulate numerous intracellular signaling pathways; often via direct interaction with specific binding partners. Caveolin-1 is particularly well-studied in this respect and has been attributed a large variety of functions. Thus, Caveolin-1 also represents the best-characterized isoform of this family with respect to its participation in cancer. Rather strikingly, available evidence indicates that Caveolin-1 belongs to a select group of proteins that function, depending on the cellular settings, both as tumor suppressor and promoter of cellular traits commonly associated with enhanced malignant behavior, such as metastasis and multi-drug resistance. The mechanisms underlying such ambiguity in Caveolin-1 function constitute an area of great interest. Here, we will focus on discussing how Caveolin-1 modulates cell death and survival pathways and how this may contribute to a better understanding of the ambiguous role this protein plays in cancer. PMID:23228128

  9. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    SciTech Connect

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing . E-mail: jnzhang@dlmedu.edu.cn

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  10. Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

    SciTech Connect

    Majkova, Zuzana; Smart, Eric; Toborek, Michal; Hennig, Bernhard

    2009-05-15

    Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R{sup -/-} mice (control) or caveolin-1{sup -/-}/LDL-R{sup -/-} mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1{sup -/-}/LDL-R{sup -/-} mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor {alpha}-naphthoflavone ({alpha}-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.

  11. Caveolin-1 as a promoter of tumour spreading: when, how, where and why

    PubMed Central

    Senetta, Rebecca; Stella, Giulia; Pozzi, Ernesto; Sturli, Niccolo; Massi, Daniela; Cassoni, Paola

    2013-01-01

    Caveolae are non-clathrin invaginations of the plasma membrane in most cell types; they are involved in signalling functions and molecule trafficking, thus modulating several biological functions, including cell growth, apoptosis and angiogenesis. The major structural protein in caveolae is caveolin-1, which is known to act as a key regulator in cancer onset and progression through its role as a tumour suppressor. Caveolin-1 can also promote cell proliferation, survival and metastasis as well as chemo- and radioresistance. Here, we discuss recent findings and novel concepts that support a role for caveolin-1 in cancer development and its distant spreading. We also address the potential application of caveolin-1 in tumour therapy and diagnosis. PMID:23521716

  12. Deceased donor multidrug resistance protein 1 and caveolin 1 gene variants may influence allograft survival in kidney transplantation

    PubMed Central

    Ma, Jun; Divers, Jasmin; Palmer, Nicholette D.; Julian, Bruce A.; Israni, Ajay K.; Schladt, David; Pastan, Stephen O.; Chattrabhuti, Kryt; Gautreaux, Michael D.; Hauptfeld, Vera; Bray, Robert A.; Kirk, Allan D.; Brown, W. Mark; Gaston, Robert S.; Rogers, Jeffrey; Farney, Alan C.; Orlando, Giuseppe; Stratta, Robert J.; Guan, Meijian; Palanisamy, Amudha; Reeves-Daniel, Amber M.; Bowden, Donald W.; Langefeld, Carl D.; Hicks, Pamela J.; Ma, Lijun; Freedman, Barry I.

    2015-01-01

    Variants in donor multidrug resistance protein 1 (ABCB1) and caveolin 1 (CAV1) genes are associated with renal allograft failure after transplantation in Europeans. Here we assessed transplantation outcomes of kidneys from 368 African American (AA) and 314 European American (EA) deceased donors based on 38 single nucleotide polymorphisms (SNPs) spanning ABCB1 and 16 SNPs spanning CAV1, including previously associated index and haplotype-tagging SNPs. Tests for association with time to allograft failure were performed for the 1,233 resultant kidney transplantations, adjusting for recipient age, sex, ethnicity, cold ischemia time, PRA, HLA match, expanded-criteria donation, and APOL1- nephropathy variants in AA donors. Interaction analyses between APOL1 with ABCB1 and CAV1 were performed. In a meta-analysis of all transplantations, ABCB1 index SNP rs1045642 was associated with time to allograft failure and other ABCB1 SNPs were nominally associated, but not CAV1 SNPs. ABCB1 SNP rs1045642 showed consistent effects with the 558 transplantations from EA donors, but not with the 675 transplantations from AA donors. ABCB1 SNP rs956825 and CAV1 SNP rs6466583 interacted with APOL1 in transplants from AA donors. Thus, the T allele at ABCB1 rs1045642 is associated with shorter renal allograft survival for kidneys from American donors. Interactions between ABCB1 and CAV1 with APOL1 may influence allograft failure for transplanted kidneys from AA donors. PMID:25853335

  13. Expression and potential correlation among Forkhead box protein M1, Caveolin-1 and E-cadherin in colorectal cancer

    PubMed Central

    Zhang, Jing; Zhang, Kundong; Zhou, Lisheng; Wu, Weidong; Jiang, Tao; Cao, Jun; Huang, Kejian; Qiu, Zhengjun; Huang, Chen

    2016-01-01

    The aim of the present study was to investigate the expression and functions of Forkhead box protein M1 (FoxM1), Caveolin-1 (Cav-1) and E-cadherin in colorectal cancer (CRC), and to determine the correlations among these proteins in CRC development and progression. The protein expression of FoxM1, Cav-1 and E-cadherin was identified using a human CRC and normal tissue microarray. A standard immunohistochemistry assay was performed employing anti-FoxM1, anti-Cav-1 and anti-E-cadherin antibodies. The clinicopathological significance of FoxM1, Cav-1 and E-cadherin in CRC was determined, and correlations were investigated between FoxM1 and Cav-1, FoxM1 and E-cadherin, Cav-1 and E-cadherin, respectively. The level of FoxM1, Cav-1 and E-Cadherin protein expression in CRC was found to be associated with pathological grade, tumor clinical stages and the presence of metastasis, respectively. Elevated expression of FoxM1 and Cav-1 was observed in the CRC tissues, and a significant correlation was found between the two proteins in CRC. However, it was also observed that FoxM1 was overexpressed while E-cadherin expression was low, indicating that there was a negative correlation between FoxM1 expression and E-cadherin expression. Moreover, there was also a negative correlation between Cav-1 and E-cadherin expression. Overall, the elevated expression of FoxM1 and Cav-1 in a human CRC microarray provided novel clinical evidence to elucidate the fact that they may play a critical role in the development and progression of CRC by negatively regulating E-cadherin expression. Furthermore, the positive correlation between FoxM1 and Cav-1 suggested that the proteins may constitute a novel signaling pathway in human CRC.

  14. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality might be due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet characterized non-estrogenic pathway. We report here that SPI-fed rat serum inhibited osteoblastic c...

  15. Caveolin-1-deficient Mice Show Accelerated Mammary Gland Development During Pregnancy, Premature Lactation, and Hyperactivation of the Jak-2/STAT5a Signaling Cascade

    PubMed Central

    Park, David S.; Lee, Hyangkyu; Frank, Philippe G.; Razani, Babak; Nguyen, Andrew V.; Parlow, Albert F.; Russell, Robert G.; Hulit, James; Pestell, Richard G.; Lisanti, Michael P.

    2002-01-01

    It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (−/−) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (−/−) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling. PMID:12388746

  16. Caveolin-1-mediated suppression of cyclooxygenase-2 via a beta-catenin-Tcf/Lef-dependent transcriptional mechanism reduced prostaglandin E2 production and survivin expression.

    PubMed

    Rodriguez, Diego A; Tapia, Julio C; Fernandez, Jaime G; Torres, Vicente A; Muñoz, Nicolas; Galleguillos, Daniela; Leyton, Lisette; Quest, Andrew F G

    2009-04-01

    Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E(2) (PGE(2)) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and beta-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE(2) and cell proliferation. Moreover, COX-2 overexpression or PGE(2) supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE(2) to the medium prevented effects attributed to caveolin-1-mediated inhibition of beta-catenin-Tcf/Lef-dependent transcription. Finally, PGE(2) reduced the coimmunoprecipitation of caveolin-1 with beta-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE(2)-induced signaling events linked to beta-catenin/Tcf/Lef-dependent transcription of tumor survival genes including cox-2 itself and survivin. PMID:19244345

  17. Caveolin-1 expression in ovarian carcinoma is MDR1 independent.

    PubMed

    Davidson, Ben; Goldberg, Iris; Givant-Horwitz, Vered; Nesland, Jahn M; Berner, Aasmund; Bryne, Magne; Risberg, Bjørn; Kopolovic, Juri; Kristensen, Gunnar B; Tropé, Claes G; van de Putte, Gregg; Reich, Reuven

    2002-02-01

    We studied the role of caveolin-1 in tumor progression and prognosis in serous ovarian carcinoma and the association between caveolin-1 and MDR1 expression. The study involved immunohistochemical analysis for caveolin-1 and P-glycoprotein (P-gp) expression in 75 effusions and 90 solid lesions from ovarian and primary peritoneal carcinoma; in situ hybridization for MDR1 messenger RNA (mRNA) expression in 62 effusions and all 90 tumors; and reverse transcription-polymerase chain reaction (RT-PCR) for caveolin-1 mRNA expression in 23 effusions. Immunohistochemical analysis localized caveolin-1 to the cell membrane in 43 effusions and 24 tumors. P-gp membrane expression was detected in 14 effusions and 11 tumors; MDR1 mRNA, in 20 effusions and 30 tumors. Caveolin-1 mRNA was expressed in 19 effusions. Caveolin-1 protein expression showed no association with that of P-gp protein or MDR1 mRNA. The expression of all markers was similar in carcinoma cells in pleural and peritoneal effusions. Caveolin-1 is a novel diagnostic marker for effusions; expression is moderately elevated in tumor cells in effusions, possibly owing to altered signal transduction and metabolism in cancer cells at this site. Expression seems MDR1 independent. PMID:11863219

  18. Mandarin Fish Caveolin 1 Interaction with Major Capsid Protein of Infectious Spleen and Kidney Necrosis Virus and Its Role in Early Stages of Infection

    PubMed Central

    Jia, Kun-Tong; Wu, Yan-Yan; Liu, Zhao-Yu; Mi, Shu; Zheng, Yi-Wen; He, Jian; Weng, Shao-Ping; Li, Shengwen Calvin

    2013-01-01

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries, particularly for mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin 1 (mCav-1) with the ISKNV major capsid protein (MCP) was detected by using a virus overlay assay and confirmed by pulldown assay and coimmunoprecipitation. This interaction was independent of the classic caveolin 1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP colocalized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as 4 h postinfection. Subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. RNA interference silencing of mCav-1 did not change virus-cell binding but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection. PMID:23283951

  19. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.

  20. Caveolin-1 overexpression in benign and malignant salivary gland tumors.

    PubMed

    Jaafari-Ashkavandi, Zohreh; Ashraf, Mohammad Javad; Nazhvani, Ali Dehghani; Azizi, Zahra

    2016-02-01

    Caveolin-1, a tyrosine-phosphorylated protein, is supposed to have different regulatory roles as promoter or suppressor in many human cancers. However, no published study concerned its expression in benign and malignant salivary gland tumors. The aim of this study was to evaluate and compare the expression of Cav-1 in the most common benign and malignant salivary gland tumors and evaluate its correlation with proliferation activity. In this cross-sectional retrospective study, immunohistochemical expression of caveolin-1 and Ki67 were evaluated in 49 samples, including 11 normal salivary glands, 15 cases of pleomorphic adenoma (PA), 13 adenoid cystic carcinomas (AdCC), and 10 mucoepidermoid carcinomas (MEC). The expression of Cav-1 was seen in 18 % of normal salivary glands and 85 % of tumors. The immunoreaction in the tumors was significantly higher than normal tissues (P = 0.001), but the difference between benign and malignant tumors was not significant (P = 0.07). Expression of Cav-1 was correlated with Ki67 labeling index in PAs, but not in malignant tumors. Cav-1 expression was not in association with tumor size and stage. Overexpression of Cav-1 was found in salivary gland tumors in comparison with normal tissues, but no significant difference was observed between benign and malignant tumors. Cav-1 was inversely correlated with proliferation in PA. Therefore, this marker may participate in tumorigenesis of salivary gland tumors and may be a potential biomarker for cancer treatments.

  1. Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

    NASA Technical Reports Server (NTRS)

    Parat, M. O.; Fox, P. L.

    2001-01-01

    Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents

  2. NS1619 regulates the expression of caveolin-1 protein in a time-dependent manner via ROS/PI3K/PKB/FoxO1 signaling pathway in brain tumor microvascular endothelial cells.

    PubMed

    Cai, Rui-Ping; Xue, Yi-Xue; Huang, Jian; Wang, Jin-Hui; Wang, Jia-Hong; Zhao, Song-Yan; Guan, Ting-Ting; Zhang, Zhou; Gu, Yan-Ting

    2016-10-15

    NS1619, a calcium-activated potassium channel (Kca channel) activator, can selectively and time-dependently accelerate the formation of transport vesicles in both the brain tumor capillary endothelium and tumor cells within 15min of treatment and then increase the permeability of the blood-brain tumor barrier (BTB). However, the mechanism involved is still under investigation. Using a rat brain glioma (C6) model, the expression of caveolin-1, FoxO1 and p-FoxO1 protein were examined at different time points after intracarotid infusion of NS1619 at a dose of 30μg/kg/min. Internalization of Cholera toxin subunit (CTB) labeled fluorescently was monitored by flow cytometry. The expression of caveolin-1 and FoxO1 protein at tumor microvessels was enhanced and caveolae-mediated CTB endocytosis was increased by NS1619 infusion for 15min. Compared with the 15min group, the expression of caveolin-1 protein was significantly decreased and the level of phosphorylation of FoxO1 was significantly increased in the NS1619 2h group. In addition, inhibitors of reactive oxygen species (ROS) or PI3K or PKB significantly attenuated the level of FoxO1 phosphorylation and also increased the expression of caveolin-1 protein in Human Brain Microvascular Endothelial Cells (HBMECs) cocultured with human glioma cells (U87) 2h after NS1619 treatment. This led to the conclusion that NS1619-mediated transport vesicle increase is, at least partly, related to the ROS/PI3K/PKB/FoxO1 signaling pathway. PMID:27653874

  3. Regulation of Phagolysosomal Digestion by Caveolin-1 of the Retinal Pigment Epithelium Is Essential for Vision.

    PubMed

    Sethna, Saumil; Chamakkala, Tess; Gu, Xiaowu; Thompson, Timothy C; Cao, Guangwen; Elliott, Michael H; Finnemann, Silvia C

    2016-03-18

    Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE). The RPE supports neighboring photoreceptors via diurnal phagocytosis of spent photoreceptor outer segment fragments. Like mice lacking caveolin-1 globally, (RPE)CAV1(-/-) mice developed a normal RPE and neural retina but showed reduced rod photoreceptor light responses, indicating that lack of caveolin-1 affects photoreceptor function in a non-cell-autonomous manner. (RPE)CAV1(-/-) RPE in situ showed normal particle engulfment but delayed phagosome clearance and reversed diurnal profiles of levels and activities of lysosomal enzymes. Therefore, eliminating caveolin-1 specifically impairs phagolysosomal degradation by the RPE in vivo. Endogenous caveolin-1 was recruited to maturing phagolysosomes in RPE cells in culture. Consistent with these in vivo data, a moderate increase (to ∼ 2.5-fold) or decrease (by half) of caveolin-1 protein levels in RPE cells in culture was sufficient to accelerate or impair phagolysosomal digestion, respectively. A mutant form of caveolin-1 that fails to reach the cell surface augmented degradation like wild-type caveolin-1. Acidic lysosomal pH and increased protease activity are essential for digestion. We show that halving caveolin-1 protein levels significantly alkalinized lysosomal pH and decreased lysosomal enzyme activities. Taken together, our results reveal a novel role for intracellular caveolin-1 in modulating phagolysosomal function. Moreover, they show, for the first time, that organellar caveolin-1 significantly affects tissue functionality in vivo. PMID:26814131

  4. Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium

    PubMed Central

    Sugiyama, Toru; Sartoretto, Juliano L.; Kalwa, Hermann; Yan, Zhonghua; Shimokawa, Hiroaki; Michel, Thomas

    2014-01-01

    Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases. PMID:24498385

  5. Loss of Caveolin 1 is Associated With the Expression of Aquaporin 1 and Bladder Dysfunction in Mice

    PubMed Central

    Jung, Seheon; Kim, Sun-Ouck; Cho, Kyung-Aa; Song, Seung Hee; Kang, Teak Won; Park, Kwangsung; Kwon, Dongdeuk

    2015-01-01

    Purpose: It is suggested that caveolin and aquaporin might be closely associated with bladder signal activity. We investigated the effect of the deletion of caveolin 1, using caveolin 1 knockout mice, on the expression of aquaporin 1 in order to identify their relation in the urothelium of the urinary bladder. Methods: The cellular localization and expressions of aquaporin 1 and caveolin 1, in the wild type and caveolin 1 knockout mice urinary bladder, were examined by Western blot and immunofluorescence techniques. Results: Aquaporin 1 and caveolin 1 were coexpressed in the arterioles, venules, and capillaries of the suburothelial layer in the wild type controls. Aquaporin 1 protein expression was significantly higher in the caveolin 1 knockout mice than in the wild type controls (P <0.05). Conclusions: The results imply that aquaporin 1 and caveolin 1 may share a distinct relation with the bladder signal activity. This might play a specific role in bladder dysfunction. PMID:25833479

  6. Caveolin-1 signaling in lung fibrosis.

    PubMed

    Tourkina, Elena; Hoffman, Stanley

    2012-01-01

    Caveolin-1 is a master regulator of several signaling cascades because it is able to bind to and thereby inhibit members of a variety of kinase families. While associated with caveolae and involved in their generation, caveolin-1 is also present at other sites. A variety of studies have suggested that caveolin-1 may be a useful therapeutic target in fibrotic diseases of the lung and other tissues because in these diseases a low level of caveolin-1 expression is associated with a high level of collagen expression and fibrosis. Reduced caveolin-1 expression is observed not only in the fibroblasts that secrete collagen, but also in epithelial cells and monocytes. This is intriguing because both epithelial cells and monocytes have been suggested to be precursors of fibroblasts. Likely downstream effects of loss of caveolin-1 in fibrosis include activation of TGF-β signaling and upregulation of CXCR4 in monocytes resulting in their enhanced migration into damaged tissue where its ligand CXCL12 is produced. Finally, it may be possible to target caveolin-1 in fibrotic diseases without the use of gene therapy. A caveolin-1 peptide (caveolin-1 scaffolding domain) has been identified that retains the function of the full-length molecule to inhibit kinases and that can be modified by addition of the Antennapedia internalization sequence to allow it to enter cells both in vitro and in vivo. PMID:22802909

  7. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway

    PubMed Central

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to 125I-albumin. HMGB1 induced an increase in 125I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  8. High Mobility Group Box Protein 1 Boosts Endothelial Albumin Transcytosis through the RAGE/Src/Caveolin-1 Pathway.

    PubMed

    Shang, Dan; Peng, Tao; Gou, Shanmiao; Li, Yiqing; Wu, Heshui; Wang, Chunyou; Yang, Zhiyong

    2016-01-01

    High-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell-cell junctions, resulting in vascular endothelial hyperpermeability. Here, we report that HMGB1 increases the endothelial transcytosis of albumin. In mouse lung vascular endothelial cells (MLVECs), HMGB1 at a concentration of 500 ng/ml or less did not harm cell-cell junctions but rapidly induced endothelial hyperpermeability to (125)I-albumin. HMGB1 induced an increase in (125)I-albumin and AlexaFluor 488-labeled albumin internalization in endocytosis assays. Depletion of receptor for advanced glycation end products (RAGE), but not TLR2 or TLR4, suppressed HMGB1-induced albumin transcytosis and endocytosis. Genetic and pharmacological destruction of lipid rafts significantly inhibited HMGB1-induced albumin endocytosis and transcytosis. HMGB1 induced the rapid phosphorylation of caveolin (Cav)-1 and Src. Either RAGE gene silencing or soluble RAGE suppressed Cav-1 Tyr14 phosphorylation and Src Tyr418 phosphorylation. The Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) blocked HMGB1-induced Cav-1 Tyr14 phosphorylation. PP2 and overexpression of Cav-1 with a T14F mutation significantly inhibited HMGB1-induced transcytosis and albumin endocytosis. Our findings suggest that HMGB1 induces the transcytosis of albumin via RAGE-dependent Src phosphorylation and Cav-1 phosphorylation. These studies revealed a new mechanism of HMGB1-induced endothelial hyperpermeability. PMID:27572515

  9. Growth suppression of MCF-7 cancer cell-derived xenografts in nude mice by caveolin-1

    SciTech Connect

    Wu Ping; Wang Xiaohui; Li Fei; Qi Baoju; Zhu Hua; Liu Shuang; Cui Yeqing; Chen Jianwen

    2008-11-07

    Caveolin-1 is an essential structural constituent of caveolae membrane domains that has been implicated in mitogenic signaling and oncogenesis. However, the exact functional role of caveolin-1 still remains controversial. In this report, utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/cav-1 cells), we demonstrate that caveolin-1 expression dramatically inhibits invasion and migration of these cells. Importantly, in vivo experiments employing xenograft tumor models demonstrated that expression of caveolin-1 results in significant growth inhibition of breast tumors. Moreover, a dramatic delay in tumor progression was observed in MCF-7/cav-1 cells as compared with MCF-7 cells. Histological analysis of tumor sections demonstrated a marked decrease in the percentage of proliferating tumor cells (Ki-67 assay) along with an increase in apoptotic tumor cells (TUNEL assay) in MCF-7/cav-1-treated animals. Our current findings provide for the first time in vivo evidence that caveolin-1 can indeed function as a tumor suppressor in human breast adenocarcinoma derived from MCF-7 cells rather than as a tumor promoter.

  10. Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

    NASA Technical Reports Server (NTRS)

    Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

    2002-01-01

    During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

  11. Dexamethasone induces caveolin-1 in vascular endothelial cells: implications for attenuated responses to VEGF.

    PubMed

    Igarashi, Junsuke; Hashimoto, Takeshi; Shoji, Kazuyo; Yoneda, Kozo; Tsukamoto, Ikuko; Moriue, Tetsuya; Kubota, Yasuo; Kosaka, Hiroaki

    2013-04-15

    Steroids exert direct actions on cardiovascular cells, although underlying molecular mechanisms remain incompletely understood. We examined if steroids modulate abundance of caveolin-1, a regulatory protein of cell-surface receptor pathways that regulates the magnitudes of endothelial response to vascular endothelial growth factor (VEGF). Dexamethasone, a synthetic glucocorticoid, induces caveolin-1 at both levels of protein and mRNA in a time- and dose-dependent manner in pharmacologically relevant concentrations in cultured bovine aortic endothelial cells. Aldosterone, a mineralocorticoid, but not the sex steroids 17β-estradiol, testosterone, or progesterone, elicits similar caveolin-1 induction. Caveolin-1 induction by dexamethasone and that by aldosterone were abrogated by RU-486, an inhibitor of glucocorticoid receptor, and by spironolactone, a mineralocorticoid receptor inhibitor, respectively. Dexamethasone attenuates VEGF-induced responses at the levels of protein kinases Akt and ERK1/2, small-G protein Rac1, nitric oxide production, and migration. When induction of caveolin-1 by dexamethasone is attenuated either by genetically by transient transfection with small interfering RNA or pharmacologically by RU-486, kinase responses to VEGF are rescued. Dexamethasone also increases expression of caveolin-1 protein in cultured human umbilical vein endothelial cells, associated with attenuated tube formation responses of these cells when cocultured with normal fibroblasts. Immunohistochemical analyses revealed that intraperitoneal injection of dexamethasone induces endothelial caveolin-1 protein in thoracic aorta and in lung artery in healthy male rats. Thus steroids functionally attenuate endothelial responses to VEGF via caveolin-1 induction at the levels of signal transduction, migration, and tube formation, identifying a novel point of cross talk between nuclear and cell-surface receptor signaling pathways. PMID:23426970

  12. Caveolin-1 as a Prognostic Marker for Local Control After Preoperative Chemoradiation Therapy in Rectal Cancer

    SciTech Connect

    Roedel, Franz Capalbo, Gianni; Roedel, Claus; Weiss, Christian

    2009-03-01

    Purpose: Caveolin-1 is a protein marker for caveolae organelles and has an essential impact on cellular signal transduction pathways (e.g., receptor tyrosine kinases, adhesion molecules, and G-protein-coupled receptors). In the present study, we investigated the expression of caveolin-1 in patients with rectal adenocarcinoma and correlated its expression pattern with the risk for disease recurrences after preoperative chemoradiation therapy (CRT) and surgical resection. Methods and Materials: Caveolin-1 mRNA and protein expression were evaluated by Affymetrix microarray analysis (n = 20) and immunohistochemistry (n = 44) on pretreatment biopsy samples of patients with locally advanced adenocarcinoma of the rectum, and were correlated with clinical and histopathologic characteristics as well as with 5-year rates of local failure and overall survival. Results: A significantly decreased median caveolin-1 intracellular mRNA level was observed in tumor biopsy samples as compared with noncancerous mucosa. Individual mRNA levels and immunohistologic staining, however, revealed an overexpression in 7 of 20 patients (35%) and 17 of 44 patients (38.6%), respectively. Based on immunohistochemical evaluation, local control rates at 5 years for patients with tumors showing low caveolin-1 expression were significantly better than for patients with high caveolin-1-expressing carcinoma cells (p = 0.05; 92%, 95% confidence interval [95% CI] = 82-102% vs. 72%, 95% CI = 49-84%). A low caveolin-1 protein expression was also significantly related to an increased overall survival rate (p = 0.05; 45%, 95% CI 16-60% vs. 82%, 95% CI = 67-97%). Conclusion: Caveolin-1 may provide a novel prognostic marker for local control and survival after preoperative CRT and surgical resection in rectal cancer.

  13. Altered Arachidonate Distribution in Macrophages from Caveolin-1 Null Mice Leading to Reduced Eicosanoid Synthesis*

    PubMed Central

    Astudillo, Alma M.; Pérez-Chacón, Gema; Meana, Clara; Balgoma, David; Pol, Albert; del Pozo, Miguel A.; Balboa, María A.; Balsinde, Jesús

    2011-01-01

    In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E2 and LTB4 production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response. PMID:21852231

  14. Effect of quercetin and its metabolite on caveolin-1 expression induced by oxidized LDL and lysophosphatidylcholine in endothelial cells

    PubMed Central

    Kamada, Chiemi; Mukai, Rie; Kondo, Akari; Sato, Shinya; Terao, Junji

    2016-01-01

    Oxidized low-density lipoprotein contributes to atherosclerotic plaque formation, and quercetin is expected to exert anti-atherosclerotic effects. We previously reported accumulation of conjugated quercetin metabolites in the aorta of rabbits fed high-cholesterol diets with quercetin glucosides, resulting in attenuation of lipid peroxidation and inhibition of lipid accumulation. Caveolin-1, a major structural protein of caveolae in vascular endothelial cells, plays a role in atherosclerosis development. Here we investigated effects of oxidized low-density lipoprotein, quercetin and its metabolite, quercetin 3-O-β-glucuronide, on caveolin-1 expression. Oxidized low-density lipoprotein significantly upregulated caveolin-1 mRNA expression. An oxidized low-density lipoprotein component, lysophosphatidylcholine, also induced expression of both caveolin-1 mRNA and protein. However, lysophosphatidylcholine did not affect the location of caveolin-1 proteins within caveolae structures. Co-treatment with quercetin or quercetin 3-O-β-glucuronide inhibited lysophosphatidylcholine-induced caveolin-1 expression. Quercetin and quercetin 3-O-β-glucuronide also suppressed expression of adhesion molecules induced by oxidized low-density lipoprotein and lysophosphatidylcholine. These results strongly suggest lysophosphatidylcholine derived from oxidized low-density lipoprotein contributes to atherosclerotic events by upregulating caveolin-1 expression, resulting in induction of adhesion molecules. Quercetin metabolites are likely to exert an anti-atherosclerotic effect by attenuating caveolin-1 expression in endothelial cells. PMID:27257344

  15. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  16. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells

    SciTech Connect

    Shatz, Maria; Lustig, Gila; Reich, Reuven; Liscovitch, Mordechai

    2010-06-10

    Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.

  17. Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells.

    PubMed

    Wehinger, Sergio; Ortiz, Rina; Díaz, María Inés; Aguirre, Adam; Valenzuela, Manuel; Llanos, Paola; Mc Master, Christopher; Leyton, Lisette; Quest, Andrew F G

    2015-05-01

    A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation. PMID:25572853

  18. Oxidative Stress-induced Inhibition of Sirt1 by Caveolin-1 Promotes p53-dependent Premature Senescence and Stimulates the Secretion of Interleukin 6 (IL-6)*

    PubMed Central

    Volonte, Daniela; Zou, Huafei; Bartholomew, Janine N.; Liu, Zhongmin; Morel, Penelope A.; Galbiati, Ferruccio

    2015-01-01

    Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Sirt1 (amino acids 310–317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6. PMID:25512378

  19. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa

    PubMed Central

    Bajmoczi, Milan; Gadjeva, Mihaela; Alper, Seth L.; Pier, Gerald B.; Golan, David E.

    2009-01-01

    Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (ΔF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH2-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial “internalization platform” involving both caveolin-1 and functional, laterally mobile CFTR. PMID:19386787

  20. Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression

    PubMed Central

    Zhao, Yong-lin; Song, Jin-ning; Ma, Xu-dong; Zhang, Bin-fei; Li, Dan-dong; Pang, Hong-gang

    2016-01-01

    Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postulated that rosiglitazone may ameliorate diffuse axonal injury via its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone significantly reduced the levels of amyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser404(p-tau (S404)), and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S404) levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury. PMID:27482223

  1. Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

    PubMed Central

    Ostermeyer, Anne G.; Ramcharan, Lynne T.; Zeng, Youchun; Lublin, Douglas M.; Brown, Deborah A.

    2004-01-01

    Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1. PMID:14709541

  2. Immunohistochemical and molecular analysis of caveolin-1 expression in canine mammary tumors.

    PubMed

    Zuccari, D A P C; Castro, R; Gavioli, A F; Mancini, U M; Frade, C S; Leonel, C

    2012-01-01

    Caveolin-1 (Cav-1) is a structural protein present in invaginations of the cell membrane. In human breast cancer, the cav-1 gene is believed to be a tumor suppressor gene associated with inhibition of tumor metastasis. However, little is known about its expression, regulation and function in canine mammary tumors. Expression levels of cav-1 were investigated using real-time PCR and immunohistochemical detection with an anti-human Cav-1 antibody. Gene expression stability of different samples was analyzed using the geNorm software. Mammary tumors from 51 female dogs were compared to normal mammary tissue from 10 female dogs. Malignant mammary cells showed a loss of Cav-1 expression by quantitative RT-PCR and weak Cav-1 staining by immunohistochemistry compared to normal mammary gland tissue. There was a significant relationship between outcome and immunostaining as well as with tumor size, indicating that caveolin subexpression has a positive predictive value and is related to higher survival and smaller tumor size. Our findings indicate that Cav-1 is a potential prognostic marker for canine mammary tumors. PMID:22370882

  3. Effect of protein deficiency on suppressor cells.

    PubMed Central

    Khorshidi, M; Mohagheghpour, N

    1979-01-01

    The effects of moderate protein deficiency on the in vitro response of spleen cells to phytohemagglutinin in A/Jax mice were studied. The response of spleen cells from protein-deficient mice to phytohemagglutinin was found to be enhanced as compared with that of cells from control animals. Since inadequate development or function of suppressor cells in the protein-deficient mice offered a possible explanation for the enhanced lymphoproliferative activity, cocultures of spleen cells from protein-deficient and control animals were tested for their responses to phytohemagglutinin. Suppression of [3H]thymidine incorporation was detected in coculture of 25% mitomycin-treated spleen cells from control animals and 75% spleen cells from protein-deficient mice. The suppressor (regulator) elements in control spleens were found to reside in the adherent cell population. PMID:313906

  4. Fluorofenidone attenuates TGF-β1-induced lung fibroblast activation via restoring the expression of caveolin-1.

    PubMed

    Liu, Jingjing; Song, Cheng; Xiao, Qiming; Hu, Gaoyun; Tao, Lijian; Meng, Jie

    2015-02-01

    Caveolin-1 plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. We previously showed that fluorofenidone (FD), a novel pyridine agent, can attenuate bleomycin-induced experimental pulmonary fibrosis and restore the production of caveolin-1. In this study, we explore mainly whether caveolin-1 plays a critical role in the anti-pulmonary fibrosis effects of FD in vitro. The normal human lung fibroblasts (NHLFs) were cultured with transforming growth factor-β1 (TGF-β1) and then were treated with FD. Subsequently, NHLFs transfected with cav-1-siRNA were treated with TGF-β1 and/or FD. The expressions of α-smooth muscle actin (α-SMA), fibronectin, collagen I, caveolin-1, phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated P38 were measured by Western blot and/or real-time polymerase chain reaction. Fluorofenidone attenuated TGF-β1-induced expressions of α-SMA, fibronectin, and collagen I; inhibited phosphorylation of ERK, JNK, and P38; and restored caveolin-1 protein expression but cannot increase caveolin-1 mRNA level in vitro. After caveolin-1 was silenced, FD could not downregulate TGF-β1-induced expressions of α-SMA, fibronectin, and collagen I or phosphorylation of ERK, JNK, and P38. These studies demonstrate that FD, a potential antifibrotic agent, may attenuate TGF-β1-induced activation of NHLFs by restoring the expression of caveolin-1.

  5. Pulmonary hypertension and metabolic syndrome: Possible connection, PPARγ and Caveolin-1

    PubMed Central

    Mathew, Rajamma

    2014-01-01

    A number of disparate diseases can lead to pulmonary hypertension (PH), a serious disorder with a high morbidity and mortality rate. Recent studies suggest that the associated metabolic dysregulation may be an important factor adversely impacting the prognosis of PH. Furthermore, metabolic syndrome is associated with vascular diseases including PH. Inflammation plays a significant role both in PH and metabolic syndrome. Adipose tissue modulates lipid and glucose metabolism, and also produces pro- and anti-inflammatory adipokines that modulate vascular function and angiogenesis, suggesting a close functional relationship between the adipose tissue and the vasculature. Both caveolin-1, a cell membrane scaffolding protein and peroxisome proliferator-activated receptor (PPAR) γ, a ligand-activated transcription factor are abundantly expressed in the endothelial cells and adipocytes. Both caveolin-1 and PPARγ modulate proliferative and anti-apoptotic pathways, cell migration, inflammation, vascular homeostasis, and participate in lipid transport, triacylglyceride synthesis and glucose metabolism. Caveolin-1 and PPARγ regulate the production of adipokines and in turn are modulated by them. This review article summarizes the roles and inter-relationships of caveolin-1, PPARγ and adipokines in PH and metabolic syndrome. PMID:25228949

  6. Caveolin-1 Orchestrates Fibroblast Growth Factor 2 Signaling Control of Angiogenesis in Placental Artery Endothelial Cell Caveolae

    PubMed Central

    Feng, Lin; Liao, Wu-xiang; Luo, Quan; Zhang, Hong-hai; Wang, Wen; Zheng, Jing; Chen, Dong-bao

    2011-01-01

    Fibroblast growth factor (FGF) receptor 1 (FGFR1) protein was expressed as the long and short as well as some truncated forms in ovine fetoplacental artery ex vivo and in vitro. Upon FGF2 stimulation, both the long and short FGFR1s were tyrosine phosphorylated and the PI3K/AKT1 and ERK1/2 pathways were activated in a concentration- and time- dependent manner in ovine fetoplacental artery endothelial (oFPAE) cells. Blockade of the PI3K/AKT1 pathway attenuated FGF2-stimulated cell proliferation and migration as well as tube formation; blockade of the ERK1/2 pathway abolished FGF2-stimulated tube formation and partially inhibited cell proliferation and did not alter cell migration. Both AKT1 and ERK1/2 were co-fractionated with caveolin-1 and activated by FGF2 in the caveolae. Disruption of caveolae by methyl-β-cyclodextrin inhibited FGF2 activation of AKT1 and ERK1/2. FGFR1 was found in the caveolae where it physically binds to caveolin-1. FGF2 stimulated dissociation of FGFR1 from caveolin-1. Downregulation of caveolin-1 significantly attenuated the FGF2-induced activation of AKT1 and ERK1/2 and inhibited FGF2-induced cell proliferation, migration and tube formation in oFPAE cells. Pretreatment with a caveolin-1 scaffolding domain peptide to mimic caveolin-1 overexpression also inhibited these FGF2-induced angiogenic responses. These data demonstrate that caveolae function as a platform for regulating FGF2-induced angiogenesis through spatiotemporally compartmentalizing FGFR1 and the AKT1 and ERK1/2 signaling modules; the major caveolar structural protein caveolin-1 interacts with FGFR1 and paradoxically regulates FGF2-induced activation of PI3K/AKT1 and ERK1/2 pathways that coordinately regulate placental angiogenesis. PMID:21830216

  7. Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

    PubMed

    Boopathi, Ettickan; Gomes, Cristiano Mendes; Goldfarb, Robert; John, Mary; Srinivasan, Vittala Gopal; Alanzi, Jaber; Malkowicz, S Bruce; Kathuria, Hasmeena; Zderic, Stephen A; Wein, Alan J; Chacko, Samuel

    2011-05-01

    Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

  8. Caveolin-1 gene knockout impairs nitrergic function in mouse small intestine

    PubMed Central

    El-Yazbi, Ahmed F; Cho, Woo-Jung; Boddy, Geoffrey; Daniel, Edwin E

    2005-01-01

    Caveolin-1 is a plasma membrane-associated protein that is responsible for caveolae formation. It plays an important role in the regulation of the function of different signaling molecules, among which are the different isoforms of nitric oxide synthase (NOS). Nitric oxide (NO) is known to be an important inhibitory mediator in the mouse gut. Caveolin-1 knockout mice (Cav1−/−) were used to examine the effect of caveolin-1 absence on the NO function in the mouse small intestine (ileum and jejunum) compared to their genetic controls and BALB/c controls. Immunohistochemical staining showed loss of caveolin-1 and NOS in the jejunal smooth muscles and myenteric plexus interstitial cells of Cajal (ICC) of Cav1−/− mice; however, nNOS immunoreactive nerves were still present in myenteric ganglia. Under nonadrenergic noncholinergic (NANC) conditions, small intestinal tissues from Cav1−/− mice relaxed to electrical field stimulation (EFS), as did tissues from control mice. Relaxation of tissues from control mice was markedly reduced by N-omega-nitro-L-arginine (10−4 M), but relaxation of Cav1−/− animals was affected much less. Also, Cav1−/− mice tissues showed reduced relaxation responses to sodium nitroprusside (100 μM) compared to controls; yet there were no significant differences in the relaxation responses to 8-bromoguanosine-3′ : 5′-cyclic monophosphate (100 μM). Apamin (10−6 M) significantly reduced relaxations to EFS in NANC conditions in Cav1−/− mice, but not in controls. The data from this study suggest that caveolin-1 gene knockout causes alterations in the smooth muscles and the ICC, leading to an impaired NO function in the mouse small intestine that could possibly be compensated by apamin-sensitive inhibitory mediators. PMID:15937515

  9. Renal caveolin-1 expression in children with unilateral ureteropelvic junction obstruction.

    PubMed

    Vallés, Patricia G; Manucha, Walter; Carrizo, Liliana; Vega Perugorria, José; Seltzer, Alicia; Ruete, Celeste

    2007-02-01

    Caveolae are plasma membrane invaginations that contain a variety of signal transduction molecules and receptors for growth factors and cytokines. This study was performed to examine the in vivo expression and localization of caveolin-1 in kidneys from 19 children who underwent surgery release of ureteropelvic junction obstruction (UPJO) in relation to renal function and degree of tubulointerstitial fibrosis. Renal biopsies were carried out at the time of surgery for obstruction release. Kidney tissue from children of similar age removed because of carcinoma was used as control. Expression of caveolin-1 at the protein level in renal tissue and urine was demonstrated in patients with technetium 99 m labeled diethylene triamine pentaacetate ((99)Tc DTPA) renal scan 28.8+/-2% and increased tubular interstitial fibrosis in seven patients at the time of obstruction release. Colocalization staining of AT(1) angiotensin II receptor with caveolin-1 in basolateral membrane of epithelial tubule cells, enhanced AT(1) messenger ribonucleic acid (mRNA) and decreased endothelial nitric oxide synthase (eNOS), were shown in these patients. In contrast, absence of association of caveolin-1 with AT(1) receptor expression in proximal and collecting tubule membranes with AT(1) receptor mRNA and eNOS mRNA expression near control were demonstrated in 12 patients, with (99)Tc DTPA renal scan 39.7+2.1% and no evidence of tubulointerstitial fibrosis. From our results, the role of caveolin-1 as a factor contributing to the severity of the tubulointerstitial process resulting from obstructive nephropathy could be suggested.

  10. Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

    PubMed Central

    Mao, Mao; Varadarajan, Sudhahar; Fukai, Tohru; Bakhshi, Farnaz R.; Chernaya, Olga; Dudley, Samuel C.; Minshall, Richard D.; Bonini, Marcelo G.

    2014-01-01

    Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation. PMID:25158065

  11. Nitroglycerin tolerance in caveolin-1 deficient mice.

    PubMed

    Mao, Mao; Varadarajan, Sudhahar; Fukai, Tohru; Bakhshi, Farnaz R; Chernaya, Olga; Dudley, Samuel C; Minshall, Richard D; Bonini, Marcelo G

    2014-01-01

    Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48-72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.

  12. Polychlorinated biphenyl-induced VCAM-1 expression is attenuated in aortic endothelial cells isolated from caveolin-1 deficient mice

    SciTech Connect

    Han, Sung Gu; Eum, Sung Yong; Toborek, Michal; Smart, Eric; Hennig, Bernhard

    2010-07-15

    Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of cardiovascular diseases such as atherosclerosis. Vascular cell adhesion molecule-1 (VCAM-1) is a critical mediator for adhesion and uptake of monocytes across the endothelium in the early stages of atherosclerosis development. The upregulation of VCAM-1 by PCBs may be dependent on functional membrane domains called caveolae. Caveolae are particularly abundant in endothelial cell membranes and involved in trafficking and signal transduction. The objective of this study was to investigate the role of caveolae in PCB-induced endothelial cell dysfunction. Primary mouse aortic endothelial cells (MAECs) isolated from caveolin-1-deficient mice and background C57BL/6 mice were treated with coplanar PCBs, such as PCB77 and PCB126. In addition, siRNA gene silencing technique was used to knockdown caveolin-1 in porcine vascular endothelial cells. In MAECs with functional caveolae, VCAM-1 protein levels were increased after exposure to both coplanar PCBs, whereas expression levels of VCAM-1 were not significantly altered in cells deficient of caveolin-1. Furthermore, PCB-induced monocyte adhesion was attenuated in caveolin-1-deficient MAECs. Similarly, siRNA silencing of caveolin-1 in porcine endothelial cells confirmed the caveolin-1-dependent VCAM-1 expression. Treatment of cells with PCB77 and PCB126 resulted in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2), and pharmacological inhibition of ERK1/2 diminished the observed PCB-induced increase in monocyte adhesion. These findings suggest that coplanar PCBs induce adhesion molecule expression, such as VCAM-1, in endothelial cells, and that this response is regulated by caveolin-1 and functional caveolae. Our data demonstrate a critical role of functional caveolae in the activation and dysfunction of endothelial cells by coplanar PCBs.

  13. Alterations of caveolin-1 expression in a mouse model of delayed cerebral vasospasm following subarachnoid hemorrhage

    PubMed Central

    Xiong, Ye; Wang, Xue-Min; Zhong, Ming; Li, Ze-Qun; Wang, Zhi; Tian, Zuo-Fu; Zheng, Kuang; Tan, Xian-Xi

    2016-01-01

    The aim of the present study was to evaluate the expression levels of caveolin-1 in the basilar artery following delayed cerebral vasospasm (DCVS) in a rat model of subarachnoid hemorrhage (SAH), in order to investigate the association between caveolin-1 and DCVS, and its potential as a treatment for DCVS of SAH. A total of 150 Sprague Dawley rats were randomly allocated into blank, saline and SAH groups. The SAH and saline groups were subdivided into days 3, 5, 7 and 14 following the establishment of the model. The murine model of SAH was established by double injection of autologous arterial blood into the cisterna magana and DCVS was detected using Bederson neurological severity scores. Hematoxylin and eosin (HE) staining was used to observe the inner perimeter of the basilar artery pipe and variations in the thickness of the basilar artery wall. Alterations in the levels of caveolin-1 protein in the basilar artery were measured using immunofluorescence and western blot analysis; whereas alterations in the mRNA expression levels of caveolin-1 were detected by reverse transcription-quantitative polymerase chain reaction. In the present study, 15 mice succumbed to SAH-induced DCVS in the day 3 (n=3), 5 (n=5) and 7 (n=2) groups. No mortality was observed in the blank control and saline groups during the process of observation in the SAH group, All mice in the SAH groups exhibited Bederson neurological severity scores ≥1; whereas no neurological impairment was detected in the blank and normal saline groups, demonstrating the success of the model. HE staining was used to assess vasospasm and the results demonstrated that the inner perimeter of the basal artery pipe decreased at day 3 in the SAH group; whereas values peaked in the day 7 group. The thickness of the basal artery wall significantly increased (P<0.05), as compared with the blank and saline groups, in which no significant alterations in the wall thickness and the inner perimeter of the basal artery pipe

  14. Flagellin-dependent TLR5/caveolin-1 as a promising immune activator in immunosenescence

    PubMed Central

    Lim, Jae Sung; Nguyen, Kim Cuc Thi; Nguyen, Chung Truong; Jang, Ik-Soon; Han, Jung Min; Fabian, Claire; Lee, Shee Eun; Rhee, Joon Haeng; Cho, Kyung A

    2015-01-01

    The age-associated decline of immune responses causes high susceptibility to infections and reduced vaccine efficacy in the elderly. However, the mechanisms underlying age-related deficits are unclear. Here, we found that the expression and signaling of flagellin (FlaB)-dependent Toll-like receptor 5 (TLR5), unlike the other TLRs, were well maintained in old macrophages, similar to young macrophages. The expression and activation of TLR5/MyD88, but not TLR4, were sensitively regulated by the upregulation of caveolin-1 in old macrophages through direct interaction. This interaction was also confirmed using macrophages from caveolin-1 or MyD88 knockout mice. Because TLR5 and caveolin-1 were well expressed in major old tissues including lung, skin, intestine, and spleen, we analyzed in vivo immune responses via a vaccine platform with FlaB as a mucosal adjuvant for the pneumococcal surface protein A (PspA) against Streptococcus pneumoniae infection in young and aged mice. The FlaB-PspA fusion protein induced a significantly higher level of PspA-specific IgG and IgA responses and demonstrated a high protective efficacy against a lethal challenge with live S. pneumoniae in aged mice. These results suggest that caveolin-1/TLR5 signaling plays a key role in age-associated innate immune responses and that FlaB-PspA stimulation of TLR5 may be a new strategy for a mucosal vaccine adjuvant against pneumococcal infection in the elderly. PMID:26223660

  15. Telmisartan regresses left ventricular hypertrophy in caveolin-1 deficient mice

    PubMed Central

    Kreiger, Marta H; Di Lorenzo, Annarita; Teutsch, Christine; Kauser, Katalin; Sessa, William C.

    2011-01-01

    The role of angiotensin II (Ang II) in promoting cardiac hypertrophy is well known, however the role of the Ang II in a spontaneous model of hypertrophy in mice lacking the protein caveolin-1 (Cav- KO) has not been explored. In this study, WT and Cav-1 KO mice were treated with angiotensin receptor blocker (ARB), telmisartan, and cardiac function assessed by echocardiography. Treatment of Cav-1 KO mice with telmisartan significantly improved cardiac function compared to age-matched, vehicle treated Cav-1 KO mice, while telmisartan did not affected cardiac function in WT mice. Both left ventricular (LV) weight to body weight ratios and LV to tibial length ratios were also reverted by telmisartan in Cav-1 KO but not WT mice. LV hypertrophy was associated with increased expression of natriuretic peptides-A and –B, β-myosin heavy chain and TGF-β and telmisartan treatment normalized the expression of these genes. Telmisartan reduced the expression of collagen genes (Col1A and Col3A) and associated perivascular fibrosis in intramyocardial vessels in Cav-1 KO mice. In conclusion, telmisartan treatment reduces indexes of cardiac hypertrophy in this unique genetic model of spontaneous LV hypertrophy. PMID:20585312

  16. Telmisartan regresses left ventricular hypertrophy in caveolin-1-deficient mice.

    PubMed

    Krieger, Marta H; Di Lorenzo, Annarita; Teutsch, Christine; Kauser, Katalin; Sessa, William C

    2010-11-01

    The role of angiotensin II (Ang II) in promoting cardiac hypertrophy is well known; however, its role in a spontaneous model of hypertrophy in mice lacking the protein caveolin-1 (Cav-1 KO) has not been explored. In this study, WT and Cav-1 KO mice were treated with angiotensin receptor blocker (ARB), telmisartan (Telm), and cardiac function was assessed by echocardiography. Treatment of Cav-1 KO mice with Telm significantly improved cardiac function compared with age-matched vehicle-treated Cav-1 KO mice, whereas Telm did not affect cardiac function in WT mice. Both left ventricular (LV) weight to body weight ratios and LV to tibial length ratios were also reverted by Telm in Cav-1 KO but not in WT mice. LV hypertrophy was associated with increased expression of natriuretic peptides A and B, β-myosin heavy chain and TGF-β, and Telm treatment normalized the expression of these genes. Telm reduced the expression of collagen genes (Col1A and Col3A) and associated perivascular fibrosis in intramyocardial vessels in Cav-1 KO mice. In conclusion, Telm treatment reduces indexes of cardiac hypertrophy in this unique genetic model of spontaneous LV hypertrophy. PMID:20585312

  17. Potential role of caveolin-1 in acetaminophen-induced hepatotoxicity

    SciTech Connect

    Gardner, Carol R.; Gray, Joshua P.; Joseph, Laurie B.; Cervelli, Jessica; Bremer, Nicole; Kim, Yunjung; Mishin, Vladimir; Laskin, Jeffrey D.; Laskin, Debra L.

    2010-05-15

    Caveolin-1 (Cav-1) is a membrane scaffolding protein, which functions to regulate intracellular compartmentalization of various signaling molecules. In the present studies, transgenic mice with a targeted disruption of the Cav-1 gene (Cav-1{sup -/-}) were used to assess the role of Cav-1 in acetaminophen-induced hepatotoxicity. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was correlated with decreased expression of Cav-1 in the liver. Acetaminophen-induced hepatotoxicity was significantly attenuated in Cav-1{sup -/-} mice, an effect that was independent of acetaminophen metabolism. Acetaminophen administration resulted in increased hepatic expression of the oxidative stress marker, lipocalin 24p3, as well as hemeoxygenase-1, but decreased glutathione and superoxide dismutase-1; no differences were noted between the genotypes suggesting that reduced toxicity in Cav-1{sup -/-} mice is not due to alterations in antioxidant defense. In wild-type mice, acetaminophen increased mRNA expression of the pro-inflammatory cytokines, interleukin-1beta, and monocyte chemoattractant protein-1 (MCP-1), as well as cyclooxygenase-2, while 15-lipoxygenase (15-LOX), which generates anti-inflammatory lipoxins, decreased. Acetaminophen-induced changes in MCP-1 and 15-LOX expression were greater in Cav-1{sup -/-} mice. Although expression of tumor necrosis factor-alpha, a potent hepatocyte mitogen, was up-regulated in the liver of Cav-1{sup -/-} mice after acetaminophen, expression of proliferating cell nuclear antigen and survivin, markers of cellular proliferation, were delayed, which may reflect the reduced need for tissue repair. Taken together, these data demonstrate that Cav-1 plays a role in promoting inflammation and toxicity during the pathogenesis of acetaminophen-induced injury.

  18. Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

    PubMed Central

    Boothe, Tobias; Lim, Gareth E.; Cen, Haoning; Skovsø, Søs; Piske, Micah; Li, Shu Nan; Nabi, Ivan R.; Gilon, Patrick; Johnson, James D.

    2016-01-01

    Objective The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. PMID:27110488

  19. A novel caveolin-1 biomarker for clinical outcome of sarcopenia.

    PubMed

    Lin, Chih-Hsueh; Lin, Cheng-Chieh; Tsai, Chia-Wen; Chang, Wen-Shin; Yang, Mei-Do; Bau, Da-Tian

    2014-01-01

    Sarcopenia, defined by the European Working Group on Sarcopenia in Older People as leading to significantly decreased muscle mass and function, contributes to increased risk of adverse health outcomes among older people. Caveolin-1 (CAV1) is a main structural protein playing a regulatory role in signaling pathways and muscle normality. However, the role of CAV1 in the development of sarcopenia is largely unknown. In this study, we aimed to investigate the contribution of CAV1 genotype to sarcopenia in a Taiwanese population. We enrolled 175 patients with sarcopenia (56 pre-sarcopenia, 63 sarcopenia and 56 severe sarcopenia) and 327 age- and gender-matched controls in this community-based case control study. The associations of six single nucleotide polymorphisms of the CAV1 gene at C521A (rs1997623), G14713A (rs3807987), G21985A (12672038), T28608A (rs3757733), T29107A (rs7804372), and G32124A (rs3807992) with sarcopenia risk were evaluated. After grouping the sarcopenia patients together, the results showed that there was a significant differential distribution among the cases and controls in their CAV1 G14713A genotype (p=0.0235), and those carrying the AG and AA genotypes had 1.65- and 1.78-fold higher odds ratios for sarcopenia compared to those with the GG genotype (95% confidence interval=1.09-2.49 and 0.96-3.31, respectively). Furthermore, the carriers with CAV1 G14713A AG or AA genotype had a higher risk for sarcopenia and severe sarcopenia, but not pre-sarcopenia, compared to those with the GG genotype. Our findings suggest that Cav1 may play a critical role in the etiology of sarcopenia, and the A allele of Cav1 G14713A may serve as an early marker for detection of sarcopenia and severe sarcopenia.

  20. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells.

    PubMed

    Colonna, Cecilia; Podestá, Ernesto J

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  1. ACTH-induced caveolin-1 tyrosine phosphorylation is related to podosome assembly in Y1 adrenal cells

    SciTech Connect

    Colonna, Cecilia . E-mail: ccolonna@fmed.uba.ar; Podesta, Ernesto J.

    2005-04-01

    Y1 adrenocortical cells respond to ACTH with a characteristic rounding-up that facilitates cAMP signaling, critical for transport of cholesterol to the mitochondria and increase in steroid secretion. We here demonstrate that caveolin-1 participates in coupling activation of protein kinase A (PKA) to the control of cell shape. ACTH/8-Br-cAMP induced reorganization of caveolin-1-positive structures in correlation with the cellular rounding-up. Concomitant with this change, there was an increase in the phosphorylation of caveolin-1 (Tyr-14) localized at focal adhesions (FA) with reorganization of FA to rounded, ringlike structures. Colocalization with phalloidin showed that phosphocaveolin is present at the edge of actin filaments and that after ACTH stimulation F-actin dots at the cell periphery become surrounded by phosphocaveolin-1. These observations along with electron microscopy studies revealed these structures as podosomes. Podosome assembly was dependent on both PKA and tyrosine kinase activities because their formation was impaired after treatment with specific inhibitors [myristoylated PKI (mPKI) or PP2, respectively] previous to ACTH/8-Br-cAMP stimulation. These results show for the first time that ACTH induces caveolin-1 phosphorylation and podosome assembly in Y1 cells and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are related to cytoskeleton dynamics.

  2. QRFP-43 inhibits lipolysis by preventing ligand-induced complex formation between perilipin A, caveolin-1, the catalytic subunit of protein kinase and hormone-sensitive lipase in 3T3-L1 adipocytes.

    PubMed

    Mulumba, Mukandila; Granata, Riccarda; Marleau, Sylvie; Ong, Huy

    2015-05-01

    QRFP (RFamide) peptides are neuropeptides involved in food intake and adiposity regulation in rodents. We have previously shown that QRFP-43 (43RFa) and QRFP-26 (26RFa) inhibited isoproterenol (ISO)-induced lipolysis in adipocytes. However, the antilipolytic signaling pathways activated by QRFP peptides have not been investigated. In the present study, 3T3-L1 adipocytes were used to identify the main pathways involved in QRFP-43 decreasing ISO-induced lipolysis. Our results show that QRFP-43 reduced ISO-induced phosphorylation of perilipin A (PLIN) and hormone-sensitive lipase (HSL) on Ser660 by 43 and 25%, respectively, but increased Akt phosphorylation by 44%. However, the inhibition of phosphodiesterase 3B (PDE3B), a regulator of lipolysis activated by Akt, did not reverse the antilipolytic effect of QRFP-43. PDE3B inhibition reversed the decrease of Ser660 HSL phosphorylation associated with QRFP-43 antilipolytic effect. QRFP-43 also prevented PKC activation and ISO-induced Src kinases activation leading to the inhibition of the caveolin-1 (CAV-1) translocation on lipid droplets. Indeed, QRFP-43 attenuated phorbol 12-myristate 13-acetate-induced lipolysis and ISO-induced extracellular signal-regulated and Src kinases by 28, 37 and 48%, respectively. The attenuation of ISO-induced lipolysis by QRFP-43 was associated with a decrease of phosphorylated Ser660 HSL, PKA-catalytic (PKA-c) subunit and CAV-1 translocation on lipid droplets by 37, 50 and 46%, respectively. The decrease in ISO-induced CAV-1 and PKA-c translocation was associated with a reduction of PLIN phosphorylation by 44% in QRFP-43-treated adipocytes. These results suggest that QRFP-43 attenuated ISO-induced lipolysis by preventing the formation of an active complex on lipid droplets and the activation of Src kinases and PKC. PMID:25677823

  3. Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture

    PubMed Central

    2010-01-01

    Background The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication. Results Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction. Conclusion As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents. PMID:20504340

  4. Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis

    PubMed Central

    Hitkova, Ivana; Yuan, Gang; Anderl, Florian; Gerhard, Markus; Kirchner, Thomas; Reu, Simone; Röcken, Christoph; Schäfer, Claus; Schmid, Roland M.; Vogelmann, Roger; Ebert, Matthias P. A.; Burgermeister, Elke

    2013-01-01

    Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells. PMID:23592983

  5. Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

    PubMed

    Hitkova, Ivana; Yuan, Gang; Anderl, Florian; Gerhard, Markus; Kirchner, Thomas; Reu, Simone; Röcken, Christoph; Schäfer, Claus; Schmid, Roland M; Vogelmann, Roger; Ebert, Matthias P A; Burgermeister, Elke

    2013-01-01

    Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells.

  6. MicroRNA 802 stimulates ROMK channels by suppressing caveolin-1.

    PubMed

    Lin, Dao-Hong; Yue, Peng; Pan, Chunyang; Sun, Peng; Wang, Wen-Hui

    2011-06-01

    Dietary potassium stimulates the surface expression of ROMK channels in the aldosterone-sensitive distal nephron, but the mechanism by which this occurs is incompletely understood. Here, a high-potassium diet increased the transcription of microRNA (miR) 802 in the cortical collecting duct in mice. In addition, high-potassium intake decreased the expression of caveolin-1, whose 3' untranslated region contains the seed sequence of miR-802. In vitro, expression of miR-802 suppressed the expression of caveolin-1, and conversely, downregulation of endogenous miR-802 increased the expression of caveolin-1. Sucrose-gradient centrifugation suggested that caveolin-1 closely associated with ROMK channels, and immunoprecipitation showed that caveolin-1 interacted with the N terminus of ROMK. Expression of caveolin-1 varied inversely with the expression of ROMK1 in the plasma membrane, and caveolin-1 inhibited ROMK1 channel activity. Removal of the clathrin-dependent endocytosis motif from ROMK1 failed to abolish the effect of caveolin-1 on ROMK1 channel activity. Last, expression of miR-802 increased ROMK1 channel activity, an effect blocked by coexpression of caveolin-1. Taken together, miR-802 mediates the stimulatory effect of a high-potassium diet on ROMK channel activity by suppressing caveolin-1 expression, which leads to increased surface expression of ROMK channels in the distal nephron. PMID:21566059

  7. [Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

    PubMed

    Beniaminov, A D; Krasnov, G S; Dmitriev, A A; Puzanov, G A; Snopok, B A; Senchenko, V N; Kashuba, V I

    2016-01-01

    Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation. PMID:27414789

  8. Caveolin-1 expression is required for the development of pulmonary emphysema through activation of the ATM-p53-p21 pathway.

    PubMed

    Volonte, Daniela; Kahkonen, Beth; Shapiro, Steven; Di, Yuanpu; Galbiati, Ferruccio

    2009-02-27

    Free radicals play a role in aging and age-related human diseases, including pulmonary emphysema. Cigarette smoke represents a source of oxidants and is considered an environmental hazard that causes pulmonary emphysema. Here, we show that caveolin-1 activates ataxia telangiectasia-mutated (ATM) after oxidative stress by sequestering the ATM inhibitor, the catalytic subunit of protein phosphatase 2A, into caveolar membranes. We demonstrate that cigarette smoke extracts promote stress-induced premature senescence in wild type but not caveolin-1 null lung fibroblasts and that caveolin-1 expression is required for activation of the ATM-p53-p21(Waf1)(/)(Cip1) pathway following stimulation with cigarette smoke extracts in vitro. In vivo studies show that caveolin-1 expression is necessary for cigarette smoking-induced senescence of lung fibroblasts and pulmonary emphysema. These findings bring new insights into the molecular mechanism underlying free radical activation of the ATM-p53 pathway and indicate that caveolin-1 is a novel therapeutic target for the treatment and/or prevention of pulmonary emphysema.

  9. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation.

  10. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation. PMID:25683686

  11. Caveolin-1 alters the pattern of cytoplasmic Ca2+ oscillations and Ca2+-dependent gene expression by enhancing leukotriene receptor desensitization.

    PubMed

    Yeh, Yi-Chun; Tang, Ming-Jer; Parekh, Anant B

    2014-06-20

    Cytoplasmic Ca(2+) oscillations constitute a widespread signaling mode and are often generated in response to stimulation of G protein-coupled receptors that activate phospholipase C. In mast cells, repetitive Ca(2+) oscillations can be evoked by modest activation of cysteinyl leukotriene type I receptors by the physiological trigger, leukotriene C4. The Ca(2+) oscillations arise from regenerative Ca(2+) release from inositol 1,4,5-trisphosphate-sensitive stores followed by Ca(2+) entry through store-operated Ca(2+) channels, and the latter selectively activate the Ca(2+)-dependent transcription factor NFAT. The cysteinyl leukotriene type I receptors desensitize through negative feedback by protein kinase C, which terminates the oscillatory Ca(2+) response. Here, we show that the scaffolding protein caveolin-1 has a profound effect on receptor-driven Ca(2+) signals and downstream gene expression. Overexpression of caveolin-1 increased receptor-phospholipase C coupling, resulting in initially larger Ca(2+) release transients of longer duration but which then ran down quickly. NFAT-activated gene expression, triggered in response to the Ca(2+) signal, was also reduced by caveolin-1. Mutagenesis studies revealed that these effects required a functional scaffolding domain within caveolin-1. Mechanistically, the increase in Ca(2+) release in the presence of caveolin-1 activated protein kinase C, which accelerated homologous desensitization of the leukotriene receptor and thereby terminated the oscillatory Ca(2+) response. Our results reveal that caveolin-1 is a bimodal regulator of receptor-dependent Ca(2+) signaling, which fine-tunes the spatial and temporal profile of the Ca(2+) rise and thereby its ability to activate the NFAT pathway.

  12. Kainic acid induces expression of caveolin-1 in activated microglia in rat brain.

    PubMed

    Takeuchi, Shigeko; Matsuda, Wakoto; Tooyama, Ikuo; Yasuhara, Osamu

    2013-01-01

    Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation. PMID:23690214

  13. Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae

    PubMed Central

    Andrews, Allison M.; Rizzo, Victor

    2016-01-01

    Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains that function as centers of signal transduction and endocytosis. Here, we tested the concept that the MP-induced pro-inflammatory phenotype shift in endothelial cells (ECs) depends on caveolae. Consistent with previous reports, MP challenge activated ECs as evidenced by upregulation of intracellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 upregulation was mediated by activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP-1) and the epidermal growth factor receptor (EGFR). This response was absent in ECs lacking caveolin-1/caveolae. To test whether caveolae-mediated endocytosis, a dynamin-2 dependent process, is a feature of the proinflammatory response, EC’s were pretreated with the dynamin-2 inhibitor dynasore. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including, EGFR phosphorylation, activation of NF-κB and upregulation of ICAM-1 expression. Thus, our results indicate that caveolae play a role in mediating the pro-inflammatory signaling pathways which lead to EC activation in response to MPs. PMID:26891050

  14. Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells.

    PubMed

    Stuart, Elizabeth S; Webley, Wilmore C; Norkin, Leonard C

    2003-07-01

    Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.

  15. Caveolin-1–dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo

    PubMed Central

    Marchiando, Amanda M.; Shen, Le; Graham, W. Vallen; Weber, Christopher R.; Schwarz, Brad T.; Austin, Jotham R.; Raleigh, David R.; Guan, Yanfang; Watson, Alastair J.M.; Montrose, Marshall H.

    2010-01-01

    Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (α) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1–dependent endocytosis of occludin to effect structural and functional tight junction regulation. PMID:20351069

  16. Cardiopulmonary bypass increases pulmonary microvascular permeability through the Src kinase pathway: Involvement of caveolin-1 and vascular endothelial cadherin

    PubMed Central

    ZHANG, JUNWEN; JIANG, ZHAOLEI; BAO, CHUNRONG; MEI, JU; ZHU, JIAQUAN

    2016-01-01

    Changes in pulmonary microvascular permeability following cardiopulmonary bypass (CPB) and the underlying mechanisms have not yet been established. Therefore, the aim of the present study was to elucidate the alterations in pulmonary microvascular permeability following CPB and the underlying mechanism. The pulmonary microvascular permeability was measured using Evans Blue dye (EBD) exclusion, and the neutrophil infiltration and proinflammatory cytokine secretion was investigated. In addition, the activation of Src kinase and the phosphorylation of caveolin-1 and vascular endothelial cadherin (VE-cadherin) was examined. The results revealed that CPB increased pulmonary microvascular leakage, neutrophil count and proinflammatory cytokines in the bronchoalveolar lavage fluid, and activated Src kinase. The administration of PP2, an inhibitor of Src kinase, decreased the activation of Src kinase and attenuated the increase in pulmonary microvascular permeability observed following CPB. Two important proteins associated with vascular permeability, caveolin-1 and VE-cadherin, were significantly activated at 24 h in the lung tissues following CPB, which correlated with the alterations in pulmonary microvascular permeability and Src kinase. PP2 administration inhibited their activation, suggesting that they are downstream factors of Src kinase activation. The data indicated that the Src kinase pathway increased pulmonary microvascular permeability following CPB, and the activation of caveolin-1 and VE-cadherin may be involved. Inhibition of this pathway may provide a potential therapy for acute lung injury following cardiac surgery. PMID:26847917

  17. Caveolin-1 affects tumor drug resistance in esophageal squamous cell carcinoma by regulating expressions of P-gp and MRP1.

    PubMed

    Zhang, Song; Cao, Wenbo; Yue, Mingjin; Zheng, Naigang; Hu, Tao; Yang, Shengli; Dong, Ziming; Lu, Shixin; Mo, Saijun

    2016-07-01

    Esophageal squamous cell carcinoma (ESCC) is the most common cancer in China, and multidrug resistance (MDR) remains one of the biggest problems in ESCC chemotherapy. In this study, we aimed to investigate the mechanism of Caveolin-1, an integral membrane protein, on regulating ESCC MDR. First, immunohistochemistry was used to check the protein expression of Caveolin-1, MDR-related protein of P-glycoprotein (P-gp), and multidrug resistance protein 1 (MRP1) in 84 pathologically characterized ESCC tissues, matched adjacent tumor, and adjacent normal-looking tissues. The results showed that Caveolin-1 expression level was elevated in ESCC tissues than that of matched adjacent tumor and adjacent normal-looking tissues (P < 0.05), and the expression of Caveolin-1 has close correlation with P-gp and MRP1 during tumor genesis of ESCC (P = 0.034, P = 0.009, respectively). Then, Caveolin-1 overexpression and knockdown were used to investigate its effect on expressions of P-gp and MRP1 in ESCC cell line Ec9706. The messenger RNA (mRNA) and protein expression levels of P-gp and MRP1 were checked by real-time quantitative reverse transcription-PCR (qRT-PCR) and Western blot (WB). The results showed that Caveolin-1 overexpression significantly promotes the mRNA and protein expression of MRP1 (P < 0.05), while almost has no effect on the mRNA and protein expression of P-gp (P > 0.05); Cavoelin-1 knockdown inhibits the mRNA and protein expressions of both P-gp and MRP1 (P < 0.05). The similar result was found in another ESCC cell line Eca109. So, it is concluded that Caveolin-1 affects ESCC MDR by regulating the expressions of P-gp and MRP1; therefore, it can be taken as a significant marker and target in tumor therapy.

  18. Caveolae and Caveolin-1 Integrate Reverse Cholesterol Transport and Inflammation in Atherosclerosis

    PubMed Central

    Qin, Li; Zhu, Neng; Ao, Bao-Xue; Liu, Chan; Shi, Ya-Ning; Du, Ke; Chen, Jian-Xiong; Zheng, Xi-Long; Liao, Duan-Fang

    2016-01-01

    Lipid disorder and inflammation play critical roles in the development of atherosclerosis. Reverse cholesterol transport is a key event in lipid metabolism. Caveolae and caveolin-1 are in the center stage of cholesterol transportation and inflammation in macrophages. Here, we propose that reverse cholesterol transport and inflammation in atherosclerosis can be integrated by caveolae and caveolin-1. PMID:27011179

  19. Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

    PubMed Central

    Gabor, Kristin A.; Kim, Dahan; Kim, Carol H.; Hess, Samuel T.

    2015-01-01

    Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale. PMID:25646724

  20. Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

    SciTech Connect

    Luo, Di-xian; Xia, Cheng-lai; Li, Jun-mu; Xiong, Yan; Yuan, Hao-yu; TANG, Zhen-Wang; Zeng, Yixin; Liao, Duan-fang

    2010-12-03

    Research highlights: {yields} Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. {yields} Static pressure induces SREBP-1 activation. {yields} Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. {yields} Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. {yields} Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 {+-} 2.8 mg/g, 31.8 {+-} 0.7 mg/g, 92.3 {+-} 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 {+-} 9.4 mg/g, 235.9 {+-} 3.0 mg/g, 386.7 {+-} 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were

  1. Lysine methylation regulates the pRb tumour suppressor protein.

    PubMed

    Munro, S; Khaire, N; Inche, A; Carr, S; La Thangue, N B

    2010-04-22

    The pRb tumour suppressor protein has a central role in coordinating early cell cycle progression. An important level of control imposed on pRb occurs through post-translational modification, for example, phosphorylation. We describe here a new level of regulation on pRb, mediated through the targeted methylation of lysine residues, by the methyltransferase Set7/9. Set7/9 methylates the C-terminal region of pRb, both in vitro and in cells, and methylated pRb interacts with heterochromatin protein HP1. pRb methylation is required for pRb-dependent cell cycle arrest and transcriptional repression, as well as pRb-dependent differentiation. Our results indicate that methylation can influence the properties of pRb, and raise the interesting possibility that methylation modulates pRb tumour suppressor activity.

  2. Altered mitochondrial function and metabolic inflexibility associated with loss of caveolin-1.

    PubMed

    Asterholm, Ingrid Wernstedt; Mundy, Dorothy I; Weng, Jian; Anderson, Richard G W; Scherer, Philipp E

    2012-02-01

    Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility. PMID:22326219

  3. Altered Mitochondrial Function and Metabolic Inflexibility Associated with Loss of Caveolin-1

    PubMed Central

    Asterholm, Ingrid Wernstedt; Mundy, Dorothy I.; Weng, Jian; Anderson, Richard G. W.; Scherer, Philipp E.

    2012-01-01

    Caveolin-1 is a major structural component of raft structures within the plasma membrane and has been implicated as a regulator of cellular signal transduction with prominent expression in adipocytes. Here, we embarked on a comprehensive characterization of the metabolic pathways dysregulated in caveolin-1 null mice. We found that these mice display decreased circulating levels of total and high molecular weight adiponectin and a reduced ability to change substrate use in response to feeding/fasting conditions. Caveolin-1 null mice are extremely lean, but retain muscle mass despite lipodystrophy and massive metabolic dysfunction. Hepatic gluconeogenesis is chronically elevated, while hepatic steatosis is reduced. Our data suggest that the complex phenotype of the caveolin-1 null mouse is caused by altered metabolic and mitochondrial function in adipose tissue with a subsequent compensatory response driven mostly by the liver. This mouse model highlights the central contributions of adipose tissue for system-wide preservation of metabolic flexibility. PMID:22326219

  4. Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression.

    PubMed

    Popa, Ioana L; Milac, Adina L; Sima, Livia E; Alexandru, Petruta R; Pastrama, Florin; Munteanu, Cristian V A; Negroiu, Gabriela

    2016-06-10

    l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

  5. Caveolin-1 is a modulator of fibroblast activation and a potential biomarker for gastric cancer.

    PubMed

    Shen, Xiao-Jun; Zhang, Hao; Tang, Gu-Sheng; Wang, Xu-Dong; Zheng, Rui; Wang, Yang; Zhu, Yan; Xue, Xu-Chao; Bi, Jian-Wei

    2015-01-01

    Stromal fibroblasts play an important role in chronic cancer-related inflammation and the development as well as progression of malignant diseases. However, the difference and relationship between inflammation-associated fibroblasts (IAFs) and cancer-associated fibroblasts (CAFs) are poorly understood. In this study, gastric cancer-associated fibroblasts (GCAFs) and their corresponding inflammation-associated fibroblasts (GIAFs) were isolated from gastric cancer (GC) with chronic gastritis and cultured in vitro. These activated fibroblasts exhibited distinct secretion and tumor-promoting behaviors in vitro. Using proteomics and bioinformatics techniques, caveolin-1 (Cav-1) was identified as a major network-centric protein of a sub-network consisting of 121 differentially expressed proteins between GIAFs and GCAFs. Furthermore, immunohistochemistry in a GC cohort showed significant difference in Cav-1 expression score between GIAFs and GCAFs and among patients with different grades of chronic gastritis. Moreover, silencing of Cav-1 in GIAFs and GCAFs using small interfering RNA increased the production of pro-inflammatory and tumor-enhancing cytokines and chemokines in conditioned mediums that elevated cell proliferation and migration when added to GC cell lines AGS and MKN45 in vitro. In addition, Cav-1 status in GIAFs and GCAFs independently predicted the prognosis of GC. Our findings indicate that Cav-1 loss contributes to the distinct activation statuses of fibroblasts in GC microenvironment and gastritis mucosa, and Cav-1 expression in both GCAFs and GIAFs may serve as a potential biomarker for GC progression. PMID:25798057

  6. Distinct structural domains of caveolin-1 independently regulate Ca2+ release-activated Ca2+ channels and Ca2+ microdomain-dependent gene expression.

    PubMed

    Yeh, Yi-Chun; Parekh, Anant B

    2015-04-01

    In eukaryotic cells, calcium entry across the cell surface activates nuclear gene expression, a process critically important for cell growth and differentiation, learning, and memory and immune cell functions. In immune cells, calcium entry occurs through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels, comprised of STIM1 and Orai1 proteins. Local calcium entry through CRAC channels activates expression of c-fos- and nuclear factor of activated T cells (NFAT)-dependent genes. Although c-fos and NFAT often interact to activate gene expression synergistically, they can be activated independently of one another to regulate distinct genes. This raises the question of how one transcription factor can be activated and not the other when both are stimulated by the same trigger. Here, we show that the lipid raft scaffolding protein caveolin-1 interacts with the STIM1-Orai1 complex to increase channel activity. Phosphorylation of tyrosine 14 on caveolin-1 regulates CRAC channel-evoked c-fos activation without impacting the NFAT pathway or Orai1 activity. Our results reveal that structurally distinct domains of caveolin-1 selectively regulate the ability of local calcium to activate distinct transcription factors. More generally, our findings reveal that modular regulation by a scaffolding protein provides a simple, yet effective, mechanism to tunnel a local signal down a specific pathway. PMID:25645930

  7. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts.

    PubMed

    Wang, Shuai; Kaartinen, Mari T

    2015-11-01

    Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9--which can alter TG enzyme conformation--localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts. PMID:26231113

  8. Oligomerization of Clostridium perfringens Epsilon Toxin Is Dependent upon Caveolins 1 and 2

    PubMed Central

    Fennessey, Christine M.; Sheng, Jinsong; Rubin, Donald H.; McClain, Mark S.

    2012-01-01

    Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2). In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1), in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization – an event which is requisite for pore formation and, by extension, cell death. PMID:23056496

  9. Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration.

    PubMed

    Cao, Jingli; Navis, Adam; Cox, Ben D; Dickson, Amy L; Gemberling, Matthew; Karra, Ravi; Bagnat, Michel; Poss, Kenneth D

    2016-01-15

    In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

  10. Altered emotionality, spatial memory and cholinergic function in caveolin-1 knock-out mice.

    PubMed

    Gioiosa, Laura; Raggi, Carla; Ricceri, Laura; Jasmin, Jean-François; Frank, Philippe G; Capozza, Franco; Lisanti, Michael P; Alleva, Enrico; Sargiacomo, Massimo; Laviola, Giovanni

    2008-04-01

    Neurological phenotypes associated with loss of caveolin 1 (cav-1) (the defining structural protein in caveolar vesicles, which regulate signal transduction and cholesterol trafficking in cells) in mice have been reported recently. In brain, cav-1 is highly expressed in neurons and glia. We investigated emotional and cognitive behavioural domains in mice deficient in cav-1 (CavKO mice). CavKO mice were more anxious and spent more time in self-directed grooming behaviour than wild-type (wt) mice. In a spatial/working memory task, CavKO mice failed to recognize the object displacement, thus showing a spatial memory impairment. CavKO mice showed higher locomotor activity than wt mice, thus suggesting reduced inhibitory function by CNS cholinergic systems. Behavioural response to the cholinergic muscarinic antagonist, scopolamine (2 mg/Kg), was decreased in CavKO mice. Few behavioural sex differences emerged in mice; whereas the sex differences were generally attenuated or even reverted in the null genotype. Our data confirm a distinct behavioural phenotype in CavKO mice and indicate a selective alteration in central cholinergic function.

  11. Pilus phase variation switches gonococcal adherence to invasion by caveolin-1-dependent host cell signaling.

    PubMed

    Faulstich, Michaela; Böttcher, Jan-Peter; Meyer, Thomas F; Fraunholz, Martin; Rudel, Thomas

    2013-01-01

    Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorB(IA), in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorB(IA)-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection. PMID:23717204

  12. Loss of caveolin-1 causes blood-retinal barrier breakdown, venous enlargement, and mural cell alteration.

    PubMed

    Gu, Xiaowu; Fliesler, Steven J; Zhao, You-Yang; Stallcup, William B; Cohen, Alex W; Elliott, Michael H

    2014-02-01

    Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

  13. PML tumor suppressor protein is required for HCV production

    SciTech Connect

    Kuroki, Misao; Ariumi, Yasuo; Hijikata, Makoto; Ikeda, Masanori; Dansako, Hiromichi; Wakita, Takaji; Shimotohno, Kunitada; Kato, Nobuyuki

    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.

  14. Protein Phosphatase-1 Inhibitor-2 Is a Novel Memory Suppressor

    PubMed Central

    Yang, Hongtian; Hou, Hailong; Pahng, Amanda; Gu, Hua; Nairn, Angus C.; Tang, Ya-Ping; Colombo, Paul J.

    2015-01-01

    Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation. SIGNIFICANCE STATEMENT We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor

  15. Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

    PubMed

    Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

    2016-10-01

    Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy. PMID:27572151

  16. Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

    PubMed

    Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

    2016-10-01

    Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy.

  17. Electroacupuncture Exerts Neuroprotection through Caveolin-1 Mediated Molecular Pathway in Intracerebral Hemorrhage of Rats

    PubMed Central

    Li, Hui-Qin; Li, Yan; Chen, Zi-Xian; Zhang, Xiao-Guang; Zheng, Xia-wei; Yang, Wen-ting; Chen, Shuang

    2016-01-01

    Spontaneous intracerebral hemorrhage (ICH) is one of the most devastating types of stroke. Here, we aim to demonstrate that electroacupuncture on Baihui (GV20) exerts neuroprotection for acute ICH possibly via the caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway. The model of ICH was established by using collagenase VII. Rats were randomly divided into three groups: Sham-operation group, Sham electroacupuncture group, and electroacupuncture group. Each group was further divided into 4 subgroups according to the time points of 6 h, 1 d, 3 d, and 7 d after ICH. The methods were used including examination of neurological deficit scores according to Longa's scale, measurement of blood-brain barrier permeability through Evans Blue content, in situ immunofluorescent detection of caveolin-1 in brains, western blot analysis of caveolin-1 in brains, and in situ zymography for measuring matrix metalloproteinase-2/9 activity in brains. Compared with Sham electroacupuncture group, electroacupuncture group has resulted in a significant improvement in neurological deficit scores and in a reduction in Evans Blue content, expression of caveolin-1, and activity of matrix metalloproteinase-2/9 at 6 h, 1 d, 3 d, and 7 d after ICH (P < 0.05). In conclusion, the present results suggested that electroacupuncture on GV20 can improve neurological deficit scores and reduce blood-brain barrier permeability after ICH, and the mechanism possibly targets caveolin-1/matrix metalloproteinase/blood-brain barrier permeability pathway. PMID:27725888

  18. Caveolin-1 Increases Proinflammatory Chemoattractants and Blood–Retinal Barrier Breakdown but Decreases Leukocyte Recruitment in Inflammation

    PubMed Central

    Li, Xiaoman; Gu, Xiaowu; Boyce, Timothy M.; Zheng, Min; Reagan, Alaina M.; Qi, Hui; Mandal, Nawajes; Cohen, Alex W.; Callegan, Michelle C.; Carr, Daniel J. J.; Elliott, Michael H.

    2014-01-01

    Purpose. Caveolin-1 (Cav-1), the signature protein of caveolae, modulates inflammatory responses, and innate immunity. However, Cav-1′s role in retinal inflammation has not been rigorously tested. In this study, we examined the effect of Cav-1 ablation on the sensitivity of the retina to inflammation. Methods. Cav-1 knockout (KO) mice were challenged by intravitreal injection of lipopolysaccharide (LPS) and inflammatory cell recruitment was assessed by flow cytometry and immunohistochemistry. Leukostasis was assessed in retinal flatmounts after perfusion with FITC-labeled Concanavalin A (FITC-ConA). Chemoattractants were measured by multiplex immunoassays. Blood–retinal barrier (BRB) breakdown was assessed quantitatively by a FITC-dextran permeability assay. The ratio of extravascular to total immune cells was determined by CD45 immunohistochemistry of retinal flatmounts. Results. Inflammatory challenge resulted in significant blunting of proinflammatory cytokine (monocyte chemoattractant protein-1 [MCP-1/CCL2], CXCL1/KC, IL-6, and IL-1β) responses as well as reduced inflammatory BRB breakdown in Cav-1 KO retinas. Paradoxically, Cav-1 deficiency resulted in significantly increased recruitment of immune cells compared with controls as well as increased leukostasis. A similar ratio of extravascular/total leukocytes were found in Cav-1 KO and wild-type (WT) retinas suggesting that Cav-1 deficient leukocytes were as competent to extravasate as those from WT mice. We found increased levels of circulating immune cells in naïve (not challenged with LPS) Cav-1 KO mice compared with controls. Conclusions. Caveolin-1 paradoxically modulates inflammatory signaling and leukocyte infiltration through distinct mechanisms. We hypothesize that Cav-1 expression may enhance inflammatory signaling while at the same time supporting the physical properties of the BRB. PMID:25159208

  19. Methamphetamine reduces expression of caveolin-1 in the dorsal striatum: Implication for dysregulation of neuronal function.

    PubMed

    Somkuwar, Sucharita S; Fannon, McKenzie J; Head, Brian P; Mandyam, Chitra D

    2016-07-22

    Role of striatal dopamine D1 receptors (D1Rs) in methamphetamine (Meth) taking and seeking is recognized from contingent Meth self-administration studies. For example, Meth increases levels of D1Rs in the dorsal striatum in animal models of Meth addiction, and blockade of striatal D1Rs decreased responding for Meth and reduced Meth priming-induced drug seeking. However, the mechanism underlying enhanced expression of striatal D1Rs in animals self-administering Meth is unknown and is hypothesized to involve maladaptive intracellular signal transduction mechanism via hyperphosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). D1Rs are predominantly localized to detergent-resistant membrane/lipid raft fractions (MLR fraction), and in vitro studies indicate that D1R signaling and recycling is regulated by the MLR-resident protein caveolin-1 (Cav-1), in an endocytotic-dependent manner. Notably, expression of Cav-1 is inversely regulated by ERK1/2 activation, suggesting a signaling interplay among D1Rs, ERK1/2 and Cav-1. We therefore evaluated the effects of extended access Meth self-administration on expression of striatal D1Rs, activated ERK1/2 and Cav-1. We first report that Cav-1 is heavily expressed in neurons located in the dorsal striatum. We also report that extended access Meth produces compulsive-like unregulated intake of the drug, and these behavioral outcomes are associated with enhanced expression of D1Rs, increased activity of ERK1/2, and reduced Cav-1 expression in the dorsal striatum. These data suggest a possible cellular mechanism that involves Cav-1 regulation of D1R expression in response to escalated Meth intake, and how this response of altered D1Rs and enhanced ERK1/2 activation to Meth self-administration contributes to contingent-related processes such as addiction. PMID:27138644

  20. Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides.

    PubMed

    Bernatchez, Pascal N; Bauer, Philip M; Yu, Jun; Prendergast, Jay S; He, Pingnian; Sessa, William C

    2005-01-18

    In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS-dependent activities. Peptides containing the Cav-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO-dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B- and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cav-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cav-1. These data highlight the importance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cav-1. PMID:15637154

  1. Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

    PubMed

    Phiboonchaiyanan, Preeyaporn Plaimee; Kiratipaiboon, Chayanin; Chanvorachote, Pithi

    2016-04-25

    Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients.

  2. Caveolin-1 Regulates Rac1 Activation and Rat Pulmonary Microvascular Endothelial Hyperpermeability Induced by TNF-α

    PubMed Central

    Sun, Geng-Yun; You, Qing-Hai; Wang, Nan; Zhang, Dan

    2013-01-01

    A multiplicity of vital cellular and tissue level functions are controlled by caveolin-1 and it is considered to be an important candidate for targeted therapeutics. Rac1-cortactin signaling plays an important role in maintaining the functions of the endothelial barrier in microvascular endothelial cells. The activity of Rac1 has been shown to be regulated by caveolin-1. Therefore, the present study investigated the consequences of down-regulating caveolin-1 and the subsequent changes in activity of Rac1 and the endothelial barrier functions in primary rat pulmonary microvascular endothelial cells (RPMVECs). RPMVECs were transfected with a small hairpin RNA duplex to down-regulate caveolin-1 expression. This procedure significantly increased the activity of Rac1. Moreover, down-regulation of caveolin-1 attenuated TNF-α-induced decrease in TER, increase in the flux of FITC-BSA and the disappearance of cortactin from the cell periphery in RPMVEC. Rac1 inhibitors significantly abolished this barrier-protective effect induced by down-regulation of caveolin-1 in response to TNF-α in RPMVECs. In conclusion, our data suggest a mechanism for the regulation of Rac1 activity by caveolin-1, with consequences for activation of endothelial cells in response to TNF-α. PMID:23383114

  3. Breast cancer nodal metastasis correlates with tumour and lymph node methylation profiles of Caveolin-1 and CXCR4.

    PubMed

    Alevizos, Leonidas; Kataki, Agapi; Derventzi, Anastasia; Gomatos, Ilias; Loutraris, Christos; Gloustianou, Georgia; Manouras, Andreas; Konstadoulakis, Manousos M; Zografos, George

    2014-06-01

    DNA methylation is the best characterised epigenetic change so far. However, its role in breast cancer metastasis has not as yet been elucidated. The aim of this study was to investigate the differences between the methylation profiles characterising primary tumours and their corresponding positive or negative for metastasis lymph nodes (LN) and correlate these with tumour metastatic potential. Methylation signatures of Caveolin-1, CXCR4, RAR-β, Cyclin D2 and Twist gene promoters were studied in 30 breast cancer primary lesions and their corresponding metastasis-free and tumour-infiltrated LN with Methylation-Specific PCR. CXCR4 and Caveolin-1 expression was further studied by immunohistochemistry. Tumours were typified by methylation of RAR-β and hypermethylation of Cyclin-D2 and Twist gene promoters. Tumour patterns were highly conserved in tumour-infiltrated LN. CXCR4 and Caveolin-1 promoter methylation patterns differentiated between node-negative and metastatic tumours. Nodal metastasis was associated with tumour and lymph node profiles of extended methylation of Caveolin-1 and lack of CXCR4 hypermethylation. Immunodetection studies verified CXCR4 and Caveolin-1 hypermethylation as gene silencing mechanism. Absence of Caveolin-1 expression in stromal cells associated with tumour aggressiveness while strong Caveolin-1 expression in tumour cells correlated with decreased 7-year disease-free survival. Methylation-mediated activation of CXCR4 and inactivation of Caveolin-1 was linked with nodal metastasis while intratumoral Caveolin-1 expression heterogeneity correlated with disease progression. This evidence contributes to the better understanding and, thereby, therapeutic management of breast cancer metastasis process.

  4. The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions

    PubMed Central

    Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J.; Infanger, Manfred; Grimm, Daniela

    2015-01-01

    We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: “NanoRacks-CellBox-Thyroid Cancer”. The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell–cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids. PMID:26633361

  5. The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions.

    PubMed

    Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J; Infanger, Manfred; Grimm, Daniela

    2015-01-01

    We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: "NanoRacks-CellBox-Thyroid Cancer". The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell-cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids. PMID:26633361

  6. Modified Panax ginseng Extract Inhibits uPAR-Mediated α[Formula: see text]β1-Integrin Signaling by Modulating Caveolin-1 to Induce Early Apoptosis in Lung Cancer Cells.

    PubMed

    Hwang, In-Hu; Kwon, Yong-Kyun; Cho, Chong-Kwan; Lee, Yeon-Weol; Sung, Jung-Suk; Joo, Jong-Cheon; Lee, Kyung-Bok; Yoo, Hwa-Seung; Jang, Ik-Soon

    2016-01-01

    Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

  7. Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.

    PubMed

    Matsumura, Shigeru; Kojidani, Tomoko; Kamioka, Yuji; Uchida, Seiichi; Haraguchi, Tokuko; Kimura, Akatsuki; Toyoshima, Fumiko

    2016-01-01

    Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry. PMID:27292265

  8. Inhibition of macrophage-derived foam cell formation by ezetimibe via the caveolin-1/MAPK pathway.

    PubMed

    Qin, Li; Yang, Yun-Bo; Yang, Yi-Xin; Zhu, Neng; Liu, Zheng; Ni, Ya-Guang; Li, Shun-Xiang; Zheng, Xi-Long; Liao, Duan-Fang

    2016-02-01

    Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol, but its effect on atherosclerosis is unclear. Foam cell formation has been implicated as a key mediator during the development of atherosclerosis. The purpose of this study was to investigate the effects of ezetimibe on foam cell formation and explore the underlying mechanism. The results presented here show that ezetimibe reduces atherosclerotic lesions in apolipoprotein E deficient (apoE-/-) mice by lowering cholesterol levels. Treatment of macrophages with Chol:MβCD resulted in foam cell formation, which was concentration-dependently inhibited by the presence of ezetimibe. Mechanically, ezetimibe treatment downregulated the expression of CD36 and scavenger receptor class B1 (SR-B1), but upregulated the expression of apoE and caveolin-1 in macrophage-derived foam cells, which kept consistent with our microarray results. Moreover, treatment with ezetimibe abrogated the increase of phospho-extracellular signal regulated kinase (ERK) 1/2 and their nuclear accumulation in foam cells. Inhibition of the MAPK pathway by the MEK inhibitor PD98059 attenuated the inhibitory effect of ezetimibe on the expression of p-ERK1/2 and caveolin-1. Taken together, these results showed that ezetimibe suppressed foam cell formation via the caveolin-1/MAPK signalling pathway, suggesting that inhibition of foam cell formation might be a novel mechanism underlying the anti-atherosclerotic effect of ezetimibe.

  9. Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1

    PubMed Central

    Matsumura, Shigeru; Kojidani, Tomoko; Kamioka, Yuji; Uchida, Seiichi; Haraguchi, Tokuko; Kimura, Akatsuki; Toyoshima, Fumiko

    2016-01-01

    Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1–LGN–NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry. PMID:27292265

  10. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    PubMed

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  11. CAVEOLIN-1 expression in brain metastasis from lung cancer predicts worse outcome and radioresistance, irrespective of tumor histotype.

    PubMed

    Duregon, Eleonora; Senetta, Rebecca; Pittaro, Alessandra; Verdun di Cantogno, Ludovica; Stella, Giulia; De Blasi, Pierpaolo; Zorzetto, Michele; Mantovani, Cristina; Papotti, Mauro; Cassoni, Paola

    2015-10-01

    Brain metastases develop in one-third of patients with non-small-cell lung cancer and are associated with a dismal prognosis, irrespective of surgery or chemo-radiotherapy. Pathological markers for predicting outcomes after surgical resection and radiotherapy responsiveness are still lacking. Caveolin 1 has been associated with chemo- and radioresistance in various tumors, including non-small-cell lung cancer. Here, caveolin 1 expression was assessed in a series of 69 brain metastases from non-small-cell lung cancer and matched primary tumors to determine its role in predicting survival and radiotherapy responsiveness. Only caveolin 1 expression in brain metastasis was associated with poor prognosis and an increased risk of death (log rank test, p = 0.015). Moreover, in the younger patients (median age of <54 years), caveolin 1 expression neutralized the favorable effect of young age on survival compared with the older patients. Among the radiotherapy-treated patients, an increased risk of death was detected in the group with caveolin 1-positive brain metastasis (14 out of 22 patients, HR=6.839, 95% CI 1.849 to 25.301, Wald test p = 0.004). Overall, caveolin 1 expression in brain metastasis from non-small-cell lung cancer is independently predictive of worse outcome and radioresistance and could become an additional tool for personalized therapy in the critical subset of brain-metastatic non-small-cell lung cancer patients.

  12. Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

    PubMed Central

    Sahoo, Anusmita; Khare, Shruti; Devanarayanan, Sivasankar; Jain, Pankaj C.; Varadarajan, Raghavan

    2015-01-01

    Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the X-ray structure being the functional one adopted in vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists. DOI: http://dx.doi.org/10.7554/eLife.09532.001 PMID:26716404

  13. Enhanced antitumor effects of low-frequency ultrasound and microbubbles in combination with simvastatin by downregulating caveolin-1 in prostatic DU145 cells

    PubMed Central

    XU, WEI-PING; SHEN, E.; BAI, WEN-KUN; WANG, YU; HU, BING

    2014-01-01

    control group (P<0.05). The expression of caveolin-1 was lowest in the LFUM combined with simvastatin treatment group. The expression of phospho-Akt (p-Akt) was consistent with caveolin-1, with the lowest expression levels of p-Akt observed in the cells that were treated with the combination of LFUM and simvastatin. The results indicate that LFUM in combination with simvastatin may additively or synergistically inhibit cell viability and induce apoptosis of DU145 cells by downregulating caveolin-1 and p-Akt protein expression. PMID:24932304

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

    SciTech Connect

    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.

  15. Caveolin-1 regulates cell apoptosis and invasion ability in paclitaxel-induced multidrug-resistant A549 lung cancer cells

    PubMed Central

    Han, Fei; Zhang, Long; Zhou, Yongxin; Yi, Xianghua

    2015-01-01

    The aim of the study was to investigate the effect and potential mechanism of caveolin-1 (Cav1) knockdown in paclitaxel-resistant lung cancer A549/Taxol cells. The human paclitaxel-resistant lung cancer cell line A549/Taxol was transfected with a Cav1 shRNA lentiviral vector. Interference efficiency for Cav1 was detected by real-time PCR and Western blotting. A MTT assay was used to determine cell proliferation, and flow cytometry was used to detect the cell cycle stage and apoptosis. Cell migration and invasion capability were detected by a transwell assay. Protein levels of related signaling molecules were detected by Western blotting. We successfully constructed a stable A549/Taxol cell line expressing low levels of Cav1. Cav1 knockdown significantly inhibited cell proliferation and induced G0/G1 arrest and cell apoptosis in vitro and in vivo. In addition, these effects correlated significantly with a reduction in cyclin D1 expression and activation of the Bcl-2/Bax-mediated mitochondrial apoptosis pathway. Furthermore, knockdown of Cav1 inhibited cell migration and invasion, and this may be related to the inhibition of AKT and the subsequent decreased protein expression of MMP2, MMP7 and MMP9. PMID:26464635

  16. DJ-1 deficiency impairs glutamate uptake into astrocytes via the regulation of flotillin-1 and caveolin-1 expression

    PubMed Central

    Kim, Jin-Mo; Cha, Seon-Heui; Choi, Yu Ree; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2016-01-01

    Parkinson’s disease (PD) is a common chronic and progressive neurodegenerative disorder. Although the cause of PD is still poorly understood, mutations in many genes including SNCA, parkin, PINK1, LRRK2, and DJ-1 have been identified in the familial forms of PD. It was recently proposed that alterations in lipid rafts may cause the neurodegeneration shown in PD. Here, we observe that DJ-1 deficiency decreased the expression of flotillin-1 (flot-1) and caveolin-1 (cav-1), the main protein components of lipid rafts, in primary astrocytes and MEF cells. As a mechanism, DJ-1 regulated flot-1 stability by direct interaction, however, decreased cav-1 expression may not be a direct effect of DJ-1, but rather as a result of decreased flot-1 expression. Dysregulation of flot-1 and cav-1 by DJ-1 deficiency caused an alteration in the cellular cholesterol level, membrane fluidity, and alteration in lipid rafts-dependent endocytosis. Moreover, DJ-1 deficiency impaired glutamate uptake into astrocytes, a major function of astrocytes in the maintenance of CNS homeostasis, by altering EAAT2 expression. This study will be helpful to understand the role of DJ-1 in the pathogenesis of PD, and the modulation of lipid rafts through the regulation of flot-1 or cav-1 may be a novel therapeutic target for PD. PMID:27346864

  17. Loss of caveolin-1 expression in knock-in mouse model of Huntington's disease suppresses pathophysiology in vivo

    PubMed Central

    Trushina, Eugenia; Canaria, Christie A.; Lee, Do-Yup; McMurray, Cynthia T.

    2014-01-01

    Loss of cholesterol homeostasis and altered vesicle trafficking have been detected in Huntington's disease (HD) cellular and animal models, yet the role of these dysfunctions in pathophysiology of HD is unknown. We demonstrate here that defects in caveolar-related cholesterol trafficking directly contribute to the mechanism of HD in vivo. We generated new mouse models that express mutant Huntington's protein (mhtt), but have partial or total loss of caveolin-1 (Cav1) expression. Fluorescence resonance energy transfer dequenching confirms a direct interaction between mhtt and Cav1. Mhtt-expressing neurons exhibited cholesterol accumulation and suppressed caveolar-related post-Golgi trafficking from endoplasmic reticulum/Golgi to plasma membrane. Loss or reduction of Cav1 expression in a knock-in HD mouse model rescues the cholesterol phenotype in neurons and significantly delays the onset of motor decline and development of neuronal inclusions. We propose that aberrant interaction between Cav1 and mhtt leads to altered cholesterol homeostasis and plays a direct causative role in the onset of HD pathophysiology in vivo. PMID:24021477

  18. Registered report: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis

    PubMed Central

    Fiering, Steven; Ang, Lay-Hong; Lacoste, Judith; Smith, Tim D; Griner, Erin; Iorns, Elizabeth

    2015-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replicating selected results 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 Altimetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from ‘Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis’ by Goetz and colleagues, published in Cell in 2011 (Goetz et al., 2011). The key experiments being replicated are those reported in Figures 7C (a-d), Supplemental Figure S2A, and Supplemental Figure S7C (a-c) (Goetz et al., 2011). In these experiments, which are a subset of all the experiments reported in the original publication, Goetz and colleagues show in a subcutaneous xenograft model that stromal caveolin-1 remodels the intratumoral microenvironment, which is correlated with increased metastasis formation. 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 in eLife. DOI: http://dx.doi.org/10.7554/eLife.04796.001 PMID:26179155

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

    PubMed Central

    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

  20. Involvement of regucalcin as a suppressor protein in human carcinogenesis: insight into the gene therapy.

    PubMed

    Yamaguchi, Masayoshi

    2015-08-01

    Regucalcin, which its gene is located on the X chromosome, plays a multifunctional role as a suppressor protein in cell signal transduction in various types of cells and tissues. The suppression of regucalcin gene expression has been shown to involve in carcinogenesis. Regucalcin gene expression was uniquely downregulated in carcinogenesis of rat liver in vivo, although the expression of other many genes was upregulated, indicating that endogenous regucalcin plays a suppressive role in the development of hepatocarcinogenesis. Overexpression of endogenous regucalcin was found to suppress proliferation of rat cloned hepatoma cells in vitro. Moreover, the regucalcin gene and its protein levels were demonstrated specifically to downregulate in human hepatocellular carcinoma by analysis with multiple gene expression profiles and proteomics. Regucalcin gene expression was also found to suppress in human tumor tissues including kidney, lung, brain, breast and prostate, suggesting that repressed regucalcin gene expression leads to the development of carcinogenesis in various tissues. Regucalcin may play a role as a suppressor protein in carcinogenesis. Overexpression of endogenous regucalcin is suggested to reveal preventive and therapeutic effects on carcinogenesis. Delivery of the regucalcin gene may be a novel useful tool in the gene therapy of carcinogenesis. This review will discuss regarding to an involvement of regucalcin as a suppressor protein in human carcinogenesis in insight into the gene therapy.

  1. Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice

    PubMed Central

    Sala-Vila, Aleix; Navarro-Lérida, Inmaculada; Sánchez-Alvarez, Miguel; Bosch, Marta; Calvo, Carlos; López, Juan Antonio; Calvo, Enrique; Ferguson, Charles; Giacomello, Marta; Serafini, Annalisa; Scorrano, Luca; Enriquez, José Antonio; Balsinde, Jesús; Parton, Robert G.; Vázquez, Jesús; Pol, Albert; Del Pozo, Miguel A.

    2016-01-01

    The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed. PMID:27272971

  2. Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice.

    PubMed

    Sala-Vila, Aleix; Navarro-Lérida, Inmaculada; Sánchez-Alvarez, Miguel; Bosch, Marta; Calvo, Carlos; López, Juan Antonio; Calvo, Enrique; Ferguson, Charles; Giacomello, Marta; Serafini, Annalisa; Scorrano, Luca; Enriquez, José Antonio; Balsinde, Jesús; Parton, Robert G; Vázquez, Jesús; Pol, Albert; Del Pozo, Miguel A

    2016-06-06

    The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed.

  3. Interplay between hepatic mitochondria-associated membranes, lipid metabolism and caveolin-1 in mice.

    PubMed

    Sala-Vila, Aleix; Navarro-Lérida, Inmaculada; Sánchez-Alvarez, Miguel; Bosch, Marta; Calvo, Carlos; López, Juan Antonio; Calvo, Enrique; Ferguson, Charles; Giacomello, Marta; Serafini, Annalisa; Scorrano, Luca; Enriquez, José Antonio; Balsinde, Jesús; Parton, Robert G; Vázquez, Jesús; Pol, Albert; Del Pozo, Miguel A

    2016-01-01

    The mitochondria-associated membrane (MAM) is a specialized subdomain of the endoplasmic reticulum (ER) which acts as an intracellular signaling hub. MAM dysfunction has been related to liver disease. We report a high-throughput mass spectrometry-based proteomics characterization of MAMs from mouse liver, which portrays them as an extremely complex compartment involved in different metabolic processes, including steroid metabolism. Interestingly, we identified caveolin-1 (CAV1) as an integral component of hepatic MAMs, which determine the relative cholesterol content of these ER subdomains. Finally, a detailed comparative proteomics analysis between MAMs from wild type and CAV1-deficient mice suggests that functional CAV1 contributes to the recruitment and regulation of intracellular steroid and lipoprotein metabolism-related processes accrued at MAMs. The potential impact of these novel aspects of CAV1 biology on global cell homeostasis and disease is discussed. PMID:27272971

  4. Caveolin-1 is overexpressed in hypopharyngeal squamous cell carcinoma and correlates with clinical parameters

    PubMed Central

    Zhao, Xuening; Yu, Gang; Yu, Xuemin; Wang, Juan; Pan, Xinliang

    2016-01-01

    The present study aimed to identify the role of caveolin-1 (CAV1) in hypopharyngeal squamous cell carcinoma (HSCC) and identify its possible correlation with tumor clinical parameters. Expression of CAV1 was measured using immunohistochemical staining of 66 HSCC samples and 44 samples from morphologically normal tissues adjacent to the carcinomas. Expression of CAV1 in HSCC and paracancerous tissues were 71.2 and 9.5% respectively. Levels of CAV1 expression were significantly associated with tumor differentiation, tumor-node-metastasis stage and lymph nodes metastasis (P<0.05). The present study identified that expression of CAV1 in HSCC is significantly higher than in paracancerous tissues, suggesting that this high expression of CAV1 is associated with tumor invasion and metastasis.

  5. Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS

    PubMed Central

    Sun, Li-na; Liu, Xiang-chun; Chen, Xiang-jun; Guan, Guang-ju; Liu, Gang

    2016-01-01

    Aim: Caveolin-1 (cav-1) is a major multifunctional scaffolding protein of caveolae. Cav-1 is primarily expressed in mesangial cells, renal proximal tubule cells and podocytes in kidneys. Recent evidence shows that the functional connections between cav-1 and ROS play a key role in many diseases. In this study we investigated whether regulating the functional connections between cav-1 and ROS in kidneys contributed to the beneficial effects of curcumin in treating diabetic nephropathy in vitro and in vivo. Methods: Cultured mouse podocytes (mpc5) were incubated in a high glucose (HG, 30 mmol/L) medium for 24, 48 or 72 h. Male rats were injected with STZ (60 mg/kg, ip) to induce diabetes. ROS generation, SOD activity, MDA content and caspase-3 activity in the cultured cells and kidney cortex homogenate were determined. Apoptotic proteins and cav-1 phosphorylation were analyzed using Western blot analyses. Results: Incubation in HG-containing medium time-dependently increased ROS production, oxidative stress, apoptosis, and cav-1 phosphorylation in podocytes. Pretreatment with curcumin (1, 5, and 10 μmol/L) dose-dependently attenuated these abnormalities in HG-treated podocytes. Furthermore, in HG-containing medium, the podocytes transfected with a recombinant plasmid GFP-cav-1 Y14F (mutation at a cav-1 phosphorylation site) exhibited significantly decreased ROS production and apoptosis compared with the cells transfected with empty vector. In diabetic rats, administration of curcumin (100 or 200 mg/kg body weight per day, ig, for 8 weeks) not only significantly improved the renal function, but also suppressed ROS levels, oxidative stress, apoptosis and cav-1 phosphorylation in the kidneys. Conclusion: Curcumin attenuates high glucose-induced podocyte apoptosis in vitro and diabetic nephropathy in vivo partly through regulating the functional connections between cav-1 phosphorylation and ROS. PMID:26838071

  6. Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

    PubMed

    Díaz, Jorge; Mendoza, Pablo; Ortiz, Rina; Díaz, Natalia; Leyton, Lisette; Stupack, Dwayne; Quest, Andrew F G; Torres, Vicente A

    2014-06-01

    Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion.

  7. Identification of caveolin-1 as a potential causative factor in the generation of trastuzumab resistance in breast cancer cells.

    PubMed

    Sekhar, Sreeja C; Kasai, Tomonari; Satoh, Ayano; Shigehiro, Tsukasa; Mizutani, Akifumi; Murakami, Hiroshi; El-Aarag, Bishoy Ya; Salomon, David S; Massaguer, Anna; de Llorens, Rafael; Seno, Masaharu

    2013-01-01

    The oncogenic tyrosine kinase receptor ErbB2 is a prognostic factor and target for breast cancer therapeutics. In contrast with the other ErbB receptors, ErbB2 is hardly internalized by ligand induced mechanisms, indicating a prevalent surface expression. Elevated levels of ErbB2 in tumor cells are associated with its defective endocytosis and down regulation. Here we show that caveolin-1 expression in breast cancer derived SKBR-3 cells (SKBR-3/Cav-1) facilitates ligand induced ErbB2 endocytosis using an artificial peptide ligand EC-eGFP. Similarly, stimulation with humanized anti ErbB2 antibody Trastuzumab (Herceptin) was found to be internalized and co-localized with caveolin-1 in SKBR-3/Cav-1 cells. Internalized EC-eGFP and Trastuzumab in SKBR-3/Cav-1 cells were then delivered via caveolae to the caveolin-1 containing early endosomes. Consequently, attenuated Fc receptor mediated ADCC functions were observed when exposed to Trastuzumab and EC-Fc (EC-1 peptide conjugated to Fc part of human IgG). On the other hand, this caveolae dependent endocytic synergy was not observed in parental SKBR-3 cells. Therefore, caveolin-1 expression in breast cancer cells could be a predictive factor to estimate how cancer cells are likely to respond to Trastuzumab treatment. PMID:23833684

  8. Developmental iodine deficiency and hypothyroidism impair neural development, upregulate caveolin-1, and downregulate synaptotagmin-1 in the rat cerebellum.

    PubMed

    Wang, Yi; Zhong, Jiapeng; Wei, Wei; Gong, Jian; Dong, Jing; Yu, Fei; Wang, Yuan; Chen, Jie

    2011-12-01

    Adequate thyroid hormone is critical for cerebellar development. Developmental hypothyroidism induced by iodine deficiency during gestation and postnatal period results in permanent impairments of cerebellar development with an unclear mechanism. In the present study, we implicate cerebellar caveolin-1 and synaptotagmin-1, the two important molecules involved in neuronal development, in developmental iodine deficiency, and in developmental hypothyroidism. Two developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil (PTU, 5 or 15 ppm)-added drinking water from gestational day 6 till postnatal day (PN) 28. Nissl staining and the levels of caveolin-1 and synaptotagmin-1 in cerebella were assessed on PN28 and PN42. The results show that the numbers of Purkinje cells were reduced in the iodine-deficient and PTU-treated rats. The upregulation of caveolin-1 and the downregulation of synaptotagmin-1 were observed in both iodine-deficient and PTU-treated rats. These findings may implicate decreases in the number of Purkinje cells and the alterations in the levels of caveolin-1 and synaptotagmin-1 in the impairments of cerebellar development induced by developmental iodine deficiency and hypothyroidism.

  9. Adherens junctional associated protein-1: a novel 1p36 tumor suppressor candidate in gliomas (Review).

    PubMed

    Zeng, Liang; Fee, Brian E; Rivas, Miriam V; Lin, James; Adamson, David Cory

    2014-07-01

    In a broad range of human cancers 1p36 has been a mutational hotspot which strongly suggests that the loss of tumor suppressor activity maps to this genomic region during tumorigenesis. Adherens junctional associated protein-1 (AJAP1; also known as Shrew1) was initially discovered as a novel transmembrane protein of adherent junctions in epithelial cells. Gene profiling showed AJAP1 on 1p36 is frequently lost or epigenetically silenced. AJAP1 may affect cell motility, migration, invasion and proliferation by unclear mechanisms. AJAP1 may be translocated to the nucleus, via its interaction with β-catenin complexes, where it can regulate gene transcription, then possibly have a potent impact on cell cycling and apoptosis. Significantly, loss of AJAP1 expression predicts poor clinical outcome of patients with malignant gliomas such as GBM and it may serve as a promising tumor suppressor-related target. In this review, we summarize and discuss current knowledge that may identify AJAP1 as a tumor suppressor in gliomas.

  10. Caveolin-1 prevents sustained angiotensin II-induced resistance artery constriction and obesity-induced high blood pressure

    PubMed Central

    Czikora, Istvan; Feher, Attila; Lucas, Rudolf; Fulton, David J. R.

    2014-01-01

    The type 1 angiotensin II (ANG II) receptor (AT1R) undergoes internalization following stimulation by ANG II. Internalization reduces cell surface AT1Rs, and it is required for AT1R resensitization. In this process AT1R may interact with caveolin-1 (Cav1), the main scaffolding protein of caveolae. We hypothesized that the interaction between Cav1 and AT1R delays AT1R resensitization and thereby prevents sustained ANG II-induced resistance artery (RA) constriction under normal conditions and in experimental obesity. In rat and mouse skeletal muscle RA (diameter: ∼90–120 μm) ANG II-induced constrictions were reduced upon repeated (30-min apart) administrations. Upon disruption of caveolae with methyl-β-cyclodextrin or in RA of Cav1 knockout mice, repeated ANG II applications resulted in essentially maintained constrictions. In vascular smooth muscle cells, AT1R interacted with Cav1, and the degree of cell surface interactions was reduced by long-term (15-min), but not short-term (2-min), exposure to ANG II. When Cav1 was silenced, the amount of membrane-associated AT1R was significantly reduced by a short-term ANG II exposure. Moreover, Cav1 knockout mice fed a high-fat diet exhibited augmented and sustained RA constriction to ANG II and had elevated systemic blood pressure, when compared with normal or high-fat fed wild-type mice. Thus, Cav1, through a direct interaction, delays internalization and subsequent resensitization of AT1R. We suggest that this mechanism prevents sustained ANG II-induced RA constriction and elevated systemic blood pressure in diet-induced obesity. PMID:25527780

  11. Overexpression of Aquaporin-1 and Caveolin-1 in the Rat Urinary Bladder Urothelium Following Bladder Outlet Obstruction

    PubMed Central

    Song, Seung Hee; Park, Kwangsung; Kwon, Dongdeuk

    2013-01-01

    Purpose This study was designed to investigate the effect of detrusor overactivity induced by partial bladder outlet obstruction (BOO) on the expression of aquaporin 1 (AQP1) and caveolin 1 (CAV1) in the rat urinary bladder, and to determine the role of these molecules in detrusor overactivity. Methods Female Sprague-Dawley rats were divided into control (n=30) and experimental (n=30) groups. The BOO group underwent partial BOO, and the control group underwent a sham operation. After 4 weeks, an urodynamic study was performed to measure the contraction interval and contraction pressure. The expression and cellular localization of AQP1 and CAV1 were determined by western blot and immunofluorescence experiments in the rat urinary bladder. Results In cystometrograms, the contraction interval was significantly lower in the BOO group (2.9±1.5 minutes) than in the control group (6.7±1.0 minutes) (P<0.05). Conversely, the average contraction pressure was significantly higher in the BOO group (21.2±3.3 mmHg) than in the control group (13.0±2.5 mmHg) (P<0.05). AQP1 and CAV1 were coexpressed in the capillaries, arterioles, and venules of the suburothelial layer. AQP1 and CAV1 protein expression was significantly increased in the BOO rats compared to the control rats (P<0.05). Conclusions Detrusor overactivity induced by BOO causes a significant increase in the expression of AQP1 and CAV1, which were coexpressed in the suburothelial microvasculature. This finding suggests that AQP1 and CAV1 might be closely related to bladder signal activity and may have a functional role in BOO-associated detrusor overactivity. PMID:24466464

  12. Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

    PubMed

    Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

    2011-03-01

    Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

  13. High Caveolin-1 Expression in Tumor Stroma Is Associated with a Favourable Outcome in Prostate Cancer Patients Managed by Watchful Waiting

    PubMed Central

    Hammarsten, Peter; Dahl Scherdin, Tove; Hägglöf, Christina; Andersson, Pernilla; Wikström, Pernilla; Stattin, Pär; Egevad, Lars; Granfors, Torvald; Bergh, Anders

    2016-01-01

    In the present study we have investigated whether Caveolin-1 expression in non-malignant and malignant prostate tissue is a potential prognostic marker for outcome in prostate cancer patients managed by watchful waiting. Caveolin-1 was measured in prostate tissues obtained through transurethral resection of the prostate from 395 patients diagnosed with prostate cancer. The majority of the patients (n = 298) were followed by watchful waiting after diagnosis. Tissue microarrays constructed from malignant and non-malignant prostate tissue were stained with an antibody against Caveolin-1. The staining pattern was scored and related to clinicopathologic parameters and outcome. Microdissection and qRT-PCR analysis of Cav-1 was done of the prostate stroma from non-malignant tissue and stroma from Gleason 3 and 4 tumors. Cav-1 RNA expression was highest in non-malignant tissue and decreased during cancer progression. High expression of Caveolin-1 in tumor stroma was associated with significantly longer cancer specific survival in prostate cancer patients. This association remained significant when Gleason score and local tumor stage were combined with Caveolin-1 in a Cox regression model. High stromal Caveolin-1 immunoreactivity in prostate tumors is associated with a favourable prognosis in prostate cancer patients managed by watchful waiting. Caveolin-1 could possibly become a useful prognostic marker for prostate cancer patients that are potential candidates for active surveillance. PMID:27764093

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

    PubMed

    Haasnoot, Joost; de Vries, Walter; Geutjes, Ernst-Jan; Prins, Marcel; de Haan, Peter; Berkhout, Ben

    2007-06-01

    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 triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication. PMID:17590081

  15. The Ebola Virus VP35 Protein Is a Suppressor of RNA Silencing

    PubMed Central

    Geutjes, Ernst-Jan; Prins, Marcel; de Haan, Peter; Berkhout, Ben

    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 triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication. PMID:17590081

  16. Hydroxylation-Dependent Interaction of Substrates to the Von Hippel-Lindau Tumor Suppressor Protein (VHL).

    PubMed

    Heir, Pardeep; Ohh, Michael

    2016-01-01

    Oxygen-dependent hydroxylation of critical proline residues, catalyzed by prolyl hydroxylase (PHD1-3) enzymes, is a crucial posttranslational modification (PTM) within the canonical hypoxia-inducible factor (HIF)-centric cellular oxygen-sensing pathway. Alteration of substrates in this way often leads to proteasomal degradation mediated by the von Hippel-Lindau Tumor Suppressor protein (VHL) containing E3-ubiquitin ligase complex known as ECV (Elongins B/C, CUL2, VHL). Here, we outline in vitro protocols to demonstrate the ability of VHL to bind to a prolyl-hydroxylated substrate. PMID:27581016

  17. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells.

    PubMed

    Lobos-González, Lorena; Aguilar, Lorena; Diaz, Jorge; Diaz, Natalia; Urra, Hery; Torres, Vicente A; Silva, Veronica; Fitzpatrick, Christopher; Lladser, Alvaro; Hoek, Keith S; Leyton, Lisette; Quest, Andrew F G

    2013-07-01

    The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

  18. Growth suppression by ursodeoxycholic acid involves caveolin-1 enhanced degradation of EGFR

    PubMed Central

    Feldman, Rebecca; Martinez, Jesse D.

    2009-01-01

    Summary Ursodeoxycholic acid (UDCA) has been shown to prevent colon tumorigenesis in animal models and in humans. In vitro work indicates that this bile acid can suppress cell growth and mitogenic signaling suggesting that UDCA may be an anti-proliferative agent. However, the mechanism by which UDCA functions is unclear. Previously we showed that bile acids may alter cellular signaling by acting at the plasma membrane. Here we utilized EGFR as a model membrane receptor and examined the effects that UDCA has on its functioning. We found that UDCA promoted an interaction between EGFR and caveolin-1 and this interaction enhanced UDCA-mediated suppression of MAP kinase activity and cell growth . Importantly, UDCA treatment led to recruitment of the ubiquitin ligase, c-Cbl, to the membrane, ubiquitination of EGFR, and increased receptor degradation. Moreover, suppression of c-Cbl activity abrogated UDCA's growth suppression activities suggesting that receptor ubiquitination plays an important role in UDCA's biological activities. Taken together these results suggest that UDCA may act to suppress cell growth by inhibiting the mitogenic activity of receptor tyrosine kinases such as EGFR through increased receptor degradation. PMID:19446582

  19. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells

    PubMed Central

    Lobos-González, L; Aguilar, L; Diaz, J; Diaz, N; Urra, H; Torres, V; Silva, V; Fitzpatrick, C; Lladser, A; Hoek, K.S.; Leyton, L; Quest, AFG

    2013-01-01

    SUMMARY The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis, and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10(cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10(E-cad) cells reduces subcutaneous tumor formation, and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10(cav1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity and cell migration observed with B16F10(cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  20. E-cadherin determines Caveolin-1 tumor suppression or metastasis enhancing function in melanoma cells.

    PubMed

    Lobos-González, Lorena; Aguilar, Lorena; Diaz, Jorge; Diaz, Natalia; Urra, Hery; Torres, Vicente A; Silva, Veronica; Fitzpatrick, Christopher; Lladser, Alvaro; Hoek, Keith S; Leyton, Lisette; Quest, Andrew F G

    2013-07-01

    The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells. PMID:23470013

  1. Neuronal Differentiation Dictates Estrogen-Dependent Survival and ERK1/2 Kinetic by Means of Caveolin-1

    PubMed Central

    Volpicelli, Floriana; Caiazzo, Massimiliano; Moncharmont, Bruno; di Porzio, Umberto; Colucci-D’Amato, Luca

    2014-01-01

    Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival. PMID:25350132

  2. Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes*

    PubMed Central

    Suzuki, Yoshiaki; Yamamura, Hisao; Ohya, Susumu; Imaizumi, Yuji

    2013-01-01

    L-type voltage-dependent Ca2+ channels (LVDCC) and large conductance Ca2+-activated K+ channels (BKCa) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca2+ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BKCa activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BKCa and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BKCa by Ca2+ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BKCa co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BKCa can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BKCa and Cav1.2 but also the functional coupling between BKCa and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K+ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BKCa-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca2+ dynamics including the negative feedback, to control the arterial excitability and contractility. PMID:24202214

  3. Modification of an apparatus for tumor-suppressor protein crystal growth in the International Space Station

    NASA Astrophysics Data System (ADS)

    de Morais Mendonca Teles, Antonio

    Some human diseases as tumors are being studied continuously for the development of vaccines against them. And a way of doing that is by means of proteins research. There are some kinds of proteins, like the p53 and p73 proteins, which are tumor suppressors. There are other diseases such as A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases which are protein-related. The determination of how proteins geometrically order themselves, during its biological functions is very necessary to understand how a protein's structure affects its function, to design vaccines that intercede in tumor-protein activities and in other proteins related to those other diseases. The protein crystal growth in microgravity environment produces purer crystallization than on the ground, and it is a powerful tool to produce better vaccines. Several data have already been acquired using ground-based research and in spaceflight experiments aboard the Spacelab and Space Shuttle missions, and in the MIR and in the International Space Station (ISS). Here in this paper, I propose to be performed in the ISS Biological Research Facility (which is being developed), multiple crystal growth of proteins related to cancer (as tumors suppressors and oncoproteins), A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases, for the future obtaining of possible vaccines against them. I also propose a simple and practical equipment, a modification of the crystallization plates (which use a vapor diffusion technique) inside each cylinder of the Protein Crystallization Apparatus in Microgravity (PCAM), with multiple chambers with different sizes. Instead of using some chambers with the same size it is better to use several chambers with different sizes. Why is that? The answer is: the energy associated with the surface tension of the liquid in the chamber is directly related to the circle area of it. So, to minimize the total energy of the surface tension of a proteins liquid -making it more stable

  4. Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

    SciTech Connect

    Luo, Di-xian; Cheng, Jiming; Xiong, Yan; Li, Junmo; Xia, Chenglai; Xu, Canxin; Wang, Chun; Zhu, Bingyang; Hu, Zhuowei; Liao, Duan-fang

    2010-01-22

    Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.

  5. The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Ross, Jonathan J; Kuzmin, Igor; Xu, Chengen; Cohen, Herbert T

    2002-10-18

    The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.

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

    SciTech Connect

    Fusaro, Adriana F.; Correa, Regis L.; Nakasugi, Kenlee; Jackson, Craig; Kawchuk, Lawrence; Vaslin, Maite F.S.; Waterhouse, Peter M.

    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.

  7. Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein

    PubMed Central

    Minervini, Giovanni; Mazzotta, Gabriella M.; Masiero, Alessandro; Sartori, Elena; Corrà, Samantha; Potenza, Emilio; Costa, Rodolfo; Tosatto, Silvio C. E.

    2015-01-01

    Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization. PMID:26211615

  8. Direct measurement of formation of loops in DNA by a human tumor suppressor protein

    NASA Astrophysics Data System (ADS)

    Migliori, Amy; Kung, Samuel; Wang, Danielle; Smith, Douglas E.

    2013-09-01

    In previous work we developed methods using optical tweezers to measure protein-mediated formation of loops in DNA structures that can play an important role in regulating gene expression. We previously applied this method to study two-site restriction endonucleases, which were convenient model systems for studying this phenomenon. Here we report preliminary work in which we have applied this method to study p53, a human tumor suppressor protein, and show that we can measure formation of loops. Previous biophysical evidence for loops comes from relatively limited qualitative studies of fixed complexes by electron microscopy4. Our results provide independent corroboration and future opportunities for more quantitative studies investigating structure and mechanics.

  9. Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

    PubMed Central

    El-Yazbi, Ahmed F; Cho, Woo Jung; Cena, Jonathan; Schulz, Richard; Daniel, Edwin E

    2008-01-01

    Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca2+] through L-type Ca2+ channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 μM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-β cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-ω-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 μM) blocked the responses to KCl and LNNA confirming the role of L-type Ca2+ channels. ODQ (1 μM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism. PMID:18400048

  10. Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

    PubMed Central

    Zimnicka, Adriana M.; Husain, Yawer S.; Shajahan, Ayesha N.; Sverdlov, Maria; Chaga, Oleg; Chen, Zhenlong; Toth, Peter T.; Klomp, Jennifer; Karginov, Andrei V.; Tiruppathi, Chinnaswamy; Malik, Asrar B.; Minshall, Richard D.

    2016-01-01

    Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or “spreading” of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane. PMID:27170175

  11. Genetic variation in caveolin-1 correlates with long-term pancreas transplant function.

    PubMed

    Hamilton, A; Mittal, S; Barnardo, M C N M; Fuggle, S V; Friend, P; Gough, S C L; Simmonds, M J

    2015-05-01

    Pancreas transplantation is a successful treatment for a selected group of people with type 1 diabetes. Continued insulin production can decrease over time and identifying predictors of long-term graft function is key to improving survival. The aim of this study was to screen subjects for variation in the Caveolin-1 gene (Cav1), previously shown to correlate with long-term kidney transplant function. We genotyped 435 pancreas transplant donors and 431 recipients who had undergone pancreas transplantation at the Oxford Transplant Centre, UK, for all known common variation in Cav1. Death-censored cumulative events were analyzed using Kaplan-Meier and Cox regression. Unlike kidney transplantation, the rs4730751 variant in our pancreas donors or transplant recipients did not correlate with long-term graft function (p = 0.331-0.905). Presence of rs3801995 TT genotype (p = 0.009) and rs9920 CC/CT genotype (p = 0.010) in our donors did however correlate with reduced long-term graft survival. Multivariate Cox regression (adjusted for donor and recipient transplant factors) confirmed the association of rs3801995 (p = 0.009, HR = 1.83;[95% CI = 1.16-2.89]) and rs9920 (p = 0.037, HR = 1.63; [95% CI = 1.03-2.73]) with long-term graft function. This is the first study to provide evidence that donor Cav1 genotype correlates with long-term pancreas graft function. Screening Cav1 in other datasets is required to confirm these pilot results.

  12. Loss of caveolin-1 and gain of MCT4 expression in the tumor stroma

    PubMed Central

    Martins, Diana; Beça, Francisco F; Sousa, Bárbara; Baltazar, Fátima; Paredes, Joana; Schmitt, Fernando

    2013-01-01

    The progression from in situ to invasive breast carcinoma is still an event poorly understood. However, it has been suggested that interactions between the neoplastic cells and the tumor microenvironment may play an important role in this process. Thus, the determination of differential tumor-stromal metabolic interactions could be an important step in invasiveness. The expression of stromal Caveolin-1 (Cav-1) has already been implicated in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Additionally, stromal Cav-1 expression has been associated with the expression of stromal monocarboxylate transporter 4 (MCT4) in invasive breast cancer. However, the role of stromal MCT4 in invasiveness has never been explored, neither the association between Cav-1 and MCT4 in the transition from breast DCIS to IDC. Therefore, our aim was to investigate in a series of breast cancer samples including matched in situ and invasive components, if there was a relationship between stromal Cav-1 and MCT4 in the progression from in situ to invasive carcinoma. We found loss of stromal Cav-1 in the progression to IDC in 75% of the cases. In contrast, MCT4 stromal expression was acquired in 87% of the IDCs. Interestingly, a concomitant loss of Cav-1 and gain of MCT4 was observed in the stroma of 75% of the cases, when matched in situ and invasive carcinomas were compared. These results suggest that alterations in Cav-1 and MCT4 may thus mark a critical point in the progression from in situ to invasive breast cancer. PMID:23907124

  13. Transmembrane adaptor protein PAG1 is a novel tumor suppressor in neuroblastoma

    PubMed Central

    Agarwal, Saurabh; Ghosh, Rajib; Chen, Zaowen; Lakoma, Anna; Gunaratne, Preethi H.; Kim, Eugene S.; Shohet, Jason M.

    2016-01-01

    (NB) is the most common extracranial pediatric solid tumor with high mortality rates. The tyrosine kinase c-Src has been known to play an important role in differentiation of NB cells, but the mechanism of c-Src regulation has not been defined. Here, we characterize PAG1 (Cbp, Csk binding protein), a central inhibitor of c-Src and other Src family kinases, as a novel tumor suppressor in NB. Clinical cohort analysis demonstrate that low expression of PAG1 is a significant prognostic factor for high stage disease, increased relapse, and worse overall survival for children with NB. PAG1 knockdown in NB cells promotes proliferation and anchorage-independent colony formation with increased activation of AKT and ERK downstream of c-Src, while PAG1 overexpression significantly rescues these effects. In vivo, PAG1 overexpression significantly inhibits NB tumorigenicity in an orthotopic xenograft model. Our results establish PAG1 as a potent tumor suppressor in NB by inhibiting c-Src and downstream effector pathways. Thus, reactivation of PAG1 and inhibition of c-Src kinase activity represents an important novel therapeutic approach for high-risk NB. PMID:26993602

  14. Bromodomain-containing protein 7 (BRD7) as a potential tumor suppressor in hepatocellular carcinoma

    PubMed Central

    Pan, Qiu-Zhong; Tang, Yan; Wang, Qi-Jing; Pan, Ke; Huang, Li-Xi; He, Jia; Zhao, Jing-Jing; Jiang, Shan-Shan; Zhang, Xiao-Fei; Zhang, Hong-Xia; Zhou, Zi-Qi; Weng, De-Sheng; Xia, Jian-Chuan

    2016-01-01

    Bromodomain-containing protein 7 (BRD7) is a subunit of the PBAF complex, which functions as a transcriptional cofactor for the tumor suppressor protein p53. Down-regulation of BRD7 has been demonstrated in multiple types of cancer. This study aimed to investigate BRD7 expression and its tumor suppressive effect in hepatocellular carcinoma (HCC). The expression of BRD7 was examined in clinical specimens of primary HCC and in HCC cell lines through real-time quantitative PCR, western blot and immunohistochemistry. The prognostic value of BRD7 expression and its correlation with the clinicopathological features of HCC patients were statistically analyzed. The effect of BRD7 on the tumorigenicity of HCC was also examined using proliferation and colony-formation assays, cell-cycle assays, migration and cell-invasion assays, and xenograft nude mouse models. BRD7 was down-regulated in tumor tissues and HCC cell lines. BRD7 protein expression was strongly associated with clinical stage and tumor size. Kaplan-Meier survival curves revealed higher survival rates in patients with higher BRD7 expression levels compared to those with lower BRD7 levels. A multivariate analysis indicated that BRD7 expression was an independent prognostic marker. The re-introduction of BRD7 expression significantly inhibited proliferation, colony formation, migration and invasion and led to cell cycle arrest in HCC cells in vitro. Furthermore, experiments in mice suggested that BRD7 overexpression suppresses HCC tumorigenicity in vivo. In conclusions, our data indicated that BRD7 may serve as a tumor suppressor in HCC and may be a novel molecular target for the treatment of HCC. PMID:26919247

  15. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy.

    PubMed

    Shi, Yin; Tan, Shi-Hao; Ng, Shukie; Zhou, Jing; Yang, Na-Di; Koo, Gi-Bang; McMahon, Kerrie-Ann; Parton, Robert G; Hill, Michelle M; Del Pozo, Miguel A; Kim, You-Sun; Shen, Han-Ming

    2015-01-01

    CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.

  16. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy

    PubMed Central

    Shi, Yin; Tan, Shi-Hao; Ng, Shukie; Zhou, Jing; Yang, Na-Di; Koo, Gi-Bang; McMahon, Kerrie-Ann; Parton, Robert G; Hill, Michelle M; del Pozo, Miguel A; Kim, You-Sun; Shen, Han-Ming

    2015-01-01

    CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy. PMID:25945613

  17. Purification and crystallization of the human EF-hand tumour suppressor protein S100A2

    PubMed Central

    Koch, Michael; Diez, Joachim; Fritz, Günter

    2006-01-01

    S100A2 is a Ca2+-binding EF-hand protein that is mainly localized in the nucleus. There, it acts as a tumour suppressor by binding and activating p53. Wild-type S100A2 and a S100A2 variant lacking cysteines have been purified. CD spectroscopy showed that there are no changes in secondary-structure composition. The S100A2 mutant was crystallized in a calcium-free form. The crystals, with dimensions 30 × 30 × 70 µm, diffract to 1.7 Å and belong to space group P212121, with unit-cell parameters a = 43.5, b = 57.8, c = 59.8 Å, α = β = γ = 90°. Preliminary analysis of the X-ray data indicates that there are two subunits per asymmetric unit. PMID:17077493

  18. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells

    PubMed Central

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi

    2015-01-01

    ABSTRACT RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic

  19. Interferon-Inducible Protein 16: Insight into the Interaction with Tumor Suppressor p53

    SciTech Connect

    Liao, Jack C.C.; Lam, Robert; Brazda, Vaclav; Duan, Shili; Ravichandran, Mani; Ma, Justin; Xiao, Ting; Tempel, Wolfram; Zuo, Xiaobing; Wang, Yun-Xing; Chirgadze, Nickolay Y.; Arrowsmith, Cheryl H.

    2011-08-24

    IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 {angstrom} resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.

  20. Stromal Caveolin-1 Is Associated With Response and Survival in a Phase II Trial of nab-Paclitaxel With Carboplatin for Advanced NSCLC Patients

    PubMed Central

    Bertino, Erin M.; Williams, Terence M.; Nana-Sinkam, S. Patrick; Shilo, Konstantin; Chatterjee, Moumita; Mo, Xiaokui; Rahmani, Meliha; Phillips, Gary S.; Villalona-Calero, Miguel A.; Otterson, Gregory A.

    2016-01-01

    In this phase II trial, carboplatin with nanoparticle albumin-bound (nab)-paclitaxel as first-line therapy for advanced non–small-cell lung cancer (NSCLC) was evaluated. Most patients had squamous cell histology. Tumor-associated stromal caveolin-1 (Cav-1) expression was correlated with improved response rate and survival in NSCLC patients who received nab-paclitaxel in this phase II trial. These results suggest Cav-1 might serve as a potential biomarker in this patient population. Background The combination of bevacizumab with platinum-based chemotherapy results in greater response rate (RR) and overall survival (OS) in advanced non–small-cell lung cancer (NSCLC). Bevacizumab is contraindicated in patients with squamous histology or hemoptysis. Nanoparticle albumin-bound (nab)-paclitaxel is a novel formulation of paclitaxel with greater dose tolerance and improved efficacy. We hypothesized that nab-paclitaxel and carboplatin would be superior to alternative doublets in advanced NSCLC patients ineligible for bevacizumab. Patients and Methods We conducted a single-arm phase II trial (NCT00729612) with carboplatin and nab-paclitaxel on day 1 of a 21-day cycle to evaluate RR (primary end point), safety, toxicity, and OS. Eligibility included: squamous histology, hemoptysis, or ongoing anticoagulation. Correlative studies included immunohistochemistry for secreted protein acid rich in cysteine (SPARC) and caveolin-1 (Cav-1). Results Sixty-three patients were enrolled. Most patients had squamous cell carcinoma (n = 48); other reasons for eligibility included hemoptysis (n = 11) and anticoagulation (n = 2). Toxicity Grade ≥ 3/4 included neuropathy, cytopenias, and fatigue. RR was 38% (24 partial response/0 complete response); 20 patients had stable disease (32%). Median progression-free survival was 5 months and median OS was 9.7 months. Immunohistochemistry for SPARC and Cav-1 was performed in 38 and 37 patients respectively. Although no association was found for

  1. Glycoproteomic Study Reveals Altered Plasma Proteins Associated with HIV Elite Suppressors

    PubMed Central

    Yang, Weiming; Laeyendecker, Oliver; Wendel, Sarah K.; Zhang, Bai; Sun, Shisheng; Zhou, Jian-Ying; Ao, Minghui; Moore, Richard D.; Jackson, J. Brooks; Zhang, Hui

    2014-01-01

    HIV elite suppressors (ES) or controllers are individuals achieving control of viremia by their natural immunological mechanisms without highly active antiretroviral therapy (HAART). Study of the mechanisms responsible for the immunological suppression of viremia in ES may lead to the detection of individuals with ES and the effective control of HIV infection. We hypothesize that plasma glycoproteins play essential roles in the immune system of ES since plasma proteins are critical and highly relevant in anti-viral immunity and most plasma proteins are glycoproteins. To examine glycoproteins associated with ES, plasma samples from ES individuals (n=20), and from individuals on HAART (n=20), with AIDS (n=20), and no HIV infection (n=10) were analyzed by quantitative glycoproteomics. We found that a number of glycoproteins changed between ES versus HAART, AIDS and HIV- individuals. In sharp contrast, the level of plasma glycoproteins in the HAART cohort showed fewer changes compared with AIDS and HIV- individuals. These results showed that although both ES and HAART effectively suppress viremia, ES appeared to profoundly affect immunologically relevant glycoproteins in plasma as consequence of or support for anti-viral immunity. Bioinformatic analysis revealed that altered proteins in ES plasma were mainly associated with inflammation. This analysis suggests that overlapping, while distinguishable, glycoprotein profiles for inflammation and immune activation appeared to be present between ES and non-ES (HAART+AIDS) cohorts, indicating different triggers for inflammation and immune activation between natural and treatment-related viral suppression. PMID:25285165

  2. Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

    PubMed Central

    Lasorella, A; Iavarone, A; Israel, M A

    1996-01-01

    Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins. PMID:8649364

  3. Super Resolution Microscopy Reveals that Caveolin-1 Is Required for Spatial Organization of CRFB1 and Subsequent Antiviral Signaling in Zebrafish

    PubMed Central

    Gabor, Kristin A.; Stevens, Chad R.; Pietraszewski, Matthew J.; Gould, Travis J.; Shim, Juyoung; Yoder, Jeffrey A.; Lam, Siew Hong; Gong, Zhiyuan; Hess, Samuel T.; Kim, Carol H.

    2013-01-01

    Understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. Caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. This study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. A novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. Viral infection leads to a downregulation in Caveolin-1b (Cav-1b), disrupting clusters of CRFB1, a zebrafish type I interferon receptor (–R) subunit. Super-resolution microscopy has enabled the first single-molecule imaging of CRFB1 association with cav-1b-containing membrane domains. Strikingly, downregulation of Cav-1b, the major protein component of caveolae, caused CRFB1 clusters to disperse. Dispersal of CRFB1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. This response strongly suggests that CRFB1 organization within cav-1b-containing membrane domains is critical for IFN-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter IFN signaling and the antiviral immune response. PMID:23874753

  4. Baicalein inhibits prostate cancer cell growth and metastasis via the caveolin-1/AKT/mTOR pathway.

    PubMed

    Guo, Zhaoxin; Hu, Xiaolin; Xing, Zhaoquan; Xing, Rui; Lv, Renguang; Cheng, Xiangyu; Su, Jing; Zhou, Zunlin; Xu, Zhonghua; Nilsson, Sten; Liu, Zhaoxu

    2015-08-01

    Prostate cancer (PCa) is lethal type of genitourinary cancer due to its high morbidity and gradual resistance to androgen deprivation therapy. Accumulating evidence has recently suggested that the daily intake of flavonoids is negatively correlated with the risk of cancer. In this study, we aimed to investigate the potential effects of baicalein on androgen-independent PCa cells and the underlying mechanisms through which baicalein exerts its actions. Cell viability and flow cytometric apoptosis assays indicated that baicalein potently suppressed the growth and induced the apoptosis of DU145 and PC-3 cells in a time- and dose-dependent manner. Consistently, the inhibitory effects of baicalein on migration and invasion were also observed in vitro. Mechanistically, we found that baicalein can suppress caveolin-1 and the phosphorylation of AKT and mTOR in a time- and dose-dependent manner. Moreover, the inhibition of the activation of AKT with LY294002 significantly promoted the apoptosis and metastasis induced by baicalein. In conclusion, these findings suggested that baicalein can induce apoptosis and inhibit metastasis of androgen-independent PCa cells through inhibition of the caveolin-1/AKT/mTOR pathway, which implies that baicalein may be a potential therapeutic agent for the treatment of androgen-independent prostate cancer patients.

  5. Heterogeneity of detergent-insoluble membranes from human intestine containing caveolin-1 and ganglioside G(M1).

    PubMed

    Badizadegan, K; Dickinson, B L; Wheeler, H E; Blumberg, R S; Holmes, R K; Lencer, W I

    2000-06-01

    In intestinal epithelia, cholera and related toxins elicit a cAMP-dependent chloride secretory response fundamental to the pathogenesis of toxigenic diarrhea. We recently proposed that specificity of cholera toxin (CT) action in model intestinal epithelia may depend on the toxin's cell surface receptor ganglioside G(M1). Binding G(M1) enabled the toxin to elicit a response, but forcing the toxin to enter the cell by binding the closely related ganglioside G(D1a) rendered the toxin inactive. The specificity of ganglioside function correlated with the ability of G(M1) to partition CT into detergent-insoluble glycosphingolipid-rich membranes (DIGs). To test the biological plausibility of these hypotheses, we examined native human intestinal epithelia. We show that human small intestinal epithelia contain DIGs that distinguish between toxin bound to G(M1) and G(D1a), thus providing a possible mechanism for enterotoxicity associated with CT. We find direct evidence for the presence of caveolin-1 in DIGs from human intestinal epithelia but find that these membranes are heterogeneous and that caveolin-1 is not a structural component of apical membrane DIGs that contain CT.

  6. 4-cholesten-3-one suppresses lung adenocarcinoma metastasis by regulating translocation of HMGB1, HIF1α and Caveolin-1

    PubMed Central

    Ma, Jinben; Fu, Guobin; Wu, Jing; Han, Shaoxian; Zhang, Lishan; Yang, Ming; Yu, Yong; Zhang, Mengyuan; Lin, Yanliang; Wang, Yibing

    2016-01-01

    Metastasis is a great challenge in lung adenocarcinoma (ADC) therapy. Cholesterol has been implicated in ADC metastasis. 4-cholesten-3-one, as cholesterol metabolite and analog, can substitute membrane cholesterol and increase membrane fluidity. In this study, we explored the possibility that 4-cholesten-3-one inhibited ADC metastasis. Low-dose 4-cholesten-3-one significantly restrained ADC cells migration and invasion with little effects on cells viabilities. Further investigation showed that 4-cholesten-3-one promoted ROS generation, which transiently activated AMPKα1, increased HIF1α expression, reduced Bcl-2 expression and caused autophagy. AMPKα1 knockdown partly suppressed 4-cholesten-3-one-induced autophagy but, neither prevented 4-cholesten-3-one-induced upregulation of HIF1α or downregulation of Bcl-2. 4-cholesten-3-one-induced autophagy facilitated the release of HMGB1 from nuclei to cytoplasm, blocking nuclear translocation of HIF1α and activation of MMP2 and MMP9. Also, 4-cholesten-3-one induced time-dependent phosphorylation of caveolin-1, Akt and NF-κB. With increasing treatment time, 4-cholesten-3-one accelerated caveolin-1 internalization, but reduced the phosphorylation of Akt and NF-κB, and inhibited the expression of snail and twist. These data suggested that 4-cholesten-3-one could be a potential candidate for anti-metastasis of lung adenocarcinoma.

  7. Negative regulation of RNA-binding protein HuR by tumor-suppressor ECRG2.

    PubMed

    Lucchesi, C; Sheikh, M S; Huang, Y

    2016-05-19

    Esophageal cancer-related gene 2 (ECRG2) is a newer tumor suppressor whose function in the regulation of cell growth and apoptosis remains to be elucidated. Here we show that ECRG2 expression was upregulated in response to DNA damage, and increased ECRG2 expression induced growth suppression in cancer cells but not in non-cancerous epithelial cells. ECRG2-mediated growth suppression was associated with activation of caspases and marked reduction in the levels of apoptosis inhibitor, X chromosome-linked inhibitor of apoptosis protein (XIAP). ECRG2, via RNA-binding protein human antigen R (HuR), regulated XIAP mRNA stability and expression. Furthermore, ECRG2 increased HuR ubiquitination and degradation but was unable to modulate the non-ubiquitinable mutant form of HuR. We also identified missense and frame-shift ECRG2 mutations in various human malignancies and noted that, unlike wild-type ECRG2, one cancer-derived ECRG2 mutant harboring glutamic acid instead of valine at position 30 (V30E) failed to induce cell death and activation of caspases. This naturally occurring V30E mutant also did not suppress XIAP and HuR. Importantly, the V30E mutant overexpressing cancer cells acquired resistance against multiple anticancer drugs, thus suggesting that ECRG2 mutations appear to have an important role in the acquisition of anticancer drug resistance in a subset of human malignancies.

  8. Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression

    PubMed Central

    Chou, Ting-Fang; Chuang, Ya-Ting; Hsieh, Wan-Chen; Chang, Pei-Yun; Liu, Hsin-Yu; Mo, Shu-Ting; Hsu, Tzu-Sheng; Miaw, Shi-Chuen; Chen, Ruey-Hwa; Kimchi, Adi; Lai, Ming-Zong

    2016-01-01

    Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK–HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk−/− Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases. PMID:27312851

  9. Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus.

    PubMed

    de Ronde, Dryas; Butterbach, Patrick; Lohuis, Dick; Hedil, Marcio; van Lent, Jan W M; Kormelink, Richard

    2013-05-01

    As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs(RI) protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N(RI) (/) (RB) protein, or when NSs(RB) was expressed. Although NSs(RI) was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs(RB) had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.

  10. Regulatory roles of tumor-suppressor proteins and noncoding RNA in cancer and normal cell functions.

    PubMed

    Garen, Alan; Song, Xu

    2008-04-15

    We describe a mechanism for reversible regulation of gene transcription, mediated by a family of tumor-suppressor proteins (TSP) containing a DNA-binding domain (DBD) that binds to a gene and represses transcription, and RNA-binding domains (RBDs) that bind RNA, usually a noncoding RNA (ncRNA), forming a TSP/RNA complex that releases the TSP from a gene and reverses repression. This mechanism appears to be involved in the regulation of embryogenesis, oncogenesis, and steroidogenesis. Embryonic cells express high levels of RNA that bind to a TSP and prevent repression of proto-oncogenes that drive cell proliferation. The level of the RNA subsequently decreases in most differentiating cells, enabling a TSP to repress proto-oncogenes and stop cell proliferation. Oncogenesis can result when the level of the RNA fails to decrease in a proliferating cell or increases in a differentiated cell. This mechanism also regulates transcription of P450scc, the first gene in the steroidogenic pathway.

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

    SciTech Connect

    Yates, Luke A. Durrant, Benjamin P.; Barber, Michael; Harlos, Karl; Fleurdépine, Sophie; Norbury, Chris J.; Gilbert, Robert J. C.

    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.

  12. Caveolin-1 is essential in the differentiation of human adipose-derived stem cells into hepatocyte-like cells via an MAPK pathway-dependent mechanism

    PubMed Central

    GUAN, XIN; WANG, NAN; CUI, FENGGONG; LIU, YANG; LIU, PENG; ZHAO, JINGYUAN; HAN, CHAO; LI, XIAOYAN; LENG, ZHIQIAN; LI, YING; JI, XIAOFEI; ZOU, WEI; LIU, JING

    2016-01-01

    Human adipose-derived stem cells (hADSCs), widely present in the adult human body, are an emerging and attractive tool for the establishment of stem cell-based therapies for the treatment of liver disease. However, the mechanism underlying hADSCs hepatic differentiation remains to be elucidated. Caveolin-1 (Cav-1), a 21–24 kDa membrane structural protein, is important in liver regeneration and development. In the present study, fluorescence immuno-cytochemistry and western blotting were used to analyze the expression levels of Cav-1 and evaluate its effects on the hepatic differentiation of hADSCs. The results revealed that primary hADSCs preserved the ability to proliferate and differentiate into hepatocyte-like cells. As demonstrated by semiquantitative reverse transcription-polymerase chain reaction, hepatocyte-inducing factors significantly increased the expression of Cav-1 in a time-dependent manner, as indicated by increased expression levels of the albumin (ALB) and α-fetoprotein (AFP) markers. In addition the expression levels of ALB and HNF1A significantly decreased following small interfering RNA-mediated knockdown of Cav-1. The mitogen-activated protein kinase (MAPK) signaling pathway was activated during hepatic differentiation and inhibited following Cav-1 knockdown. These results suggested that Cav-1 may regulate the hepatocyte-like differentiation of hADSCs by modulating mitogen-activated protein kinase kinase/MAPK signaling. The results of the present study will provide experimental and theoretical basis for further clinical studies on stem cell transplantation in the treatment of liver disease. PMID:26717806

  13. Potential RNA Binding Proteins in Saccharomyces Cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in Npl3

    PubMed Central

    Henry, M.; Borland, C. Z.; Bossie, M.; Silver, P. A.

    1996-01-01

    The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Np13p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism. PMID:8770588

  14. Restoration of caveolin-1 expression suppresses growth, membrane-type-4 metalloproteinase expression and metastasis-associated activities in colon cancer cells.

    PubMed

    Nimri, Lili; Barak, Hossei; Graeve, Lutz; Schwartz, Betty

    2013-11-01

    Caveolin-1 (cav-1) and flotillin-1 are two major structural proteins associated with lipid rafts in mammalian cells. The membrane-type matrix metalloproteinases (MT-MMPs) are expressed at the cell surface, hydrolyze extracellular matrix, and play an important role in cancer cell migration and metastasis. Expression of cav-1, flotillin-1, and MT4-MMP in lysates and lipid rafts of LS174T and HM-7 colon cancer cells was determined. The impact of restoration of cav-1 expression on proliferation, adhesion, motility in vitro, and growth of implanted tumors in vivo was characterized. Cav-1 is not expressed in lipid rafts of the highly metastatic colon cancer cell line (HM-7), but expressed in cytosolic fractions of the parental lower metastatic cell line (LS174T). In contrast, MT4-MMP was expressed in lipid rafts of HM-7 cells but not in LS174T cells. Overexpression of cav-1 in HM-7 cells down-regulate proliferation, viability, wound closure, adhesion to laminin, invasion, and development of filopodial and lamellipodial structures in a dose-dependent manner. Cav-1 positive HM-7 clones ceased to express MT4-MMP in their lipid rafts. Comparative proteomic analyses of lipid rafts from cav-1 positive and cav-1 negative cells demonstrated de novo expression of flotillin-1 only on the cells expressing cav-1. Xenografting control cells devoid of cav-1 in nude mice induced development of bigger tumors expressing higher levels of proliferating cell nuclear antigen as compared to mice injected with cells expressing the highest cav-1 levels. We conclude that cav-1 orchestrates and reorganize several proteins in lipid rafts, activities directly associated with reduced tumorigenic and metastatic ability of colon cancer cells.

  15. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage.

    PubMed

    Liu, Jie; Jin, Xinchun; Liu, Ke J; Liu, Wenlan

    2012-02-29

    Blood-brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here, we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran and promoted the secretion of metalloproteinase-2 and -9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT (2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane) or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity, and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2 h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.

  16. Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood-brain barrier damage in early ischemic stroke stage.

    PubMed

    Liu, Jie; Jin, Xinchun; Liu, Ke J; Liu, Wenlan

    2012-02-29

    Blood-brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here, we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran and promoted the secretion of metalloproteinase-2 and -9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT (2-[[(4-phenoxyphenyl)sulfonyl]methyl]-thiirane) or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity, and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2 h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis. PMID:22378877

  17. Increase in caveolae and caveolin-1 expression modulates agonist-induced contraction and store- and receptor-operated Ca(2+) entry in pulmonary arteries of pulmonary hypertensive rats.

    PubMed

    Jiao, Hai-Xia; Mu, Yun-Ping; Gui, Long-Xin; Yan, Fu-Rong; Lin, Da-Cen; Sham, James S K; Lin, Mo-Jun

    2016-09-01

    Caveolin-1 (Cav-1) is a major component protein associated with caveolae in the plasma membrane and has been identified as a regulator of store-operated Ca(2+) entry (SOCE) and receptor-operated Ca(2+) entry (ROCE). However, the contributions of caveolae/Cav-1 of pulmonary arterial smooth muscle cells (PASMCs) to the altered Ca(2+) signaling pathways in pulmonary arteries (PAs) during pulmonary hypertension (PH) have not been fully characterized. The present study quantified caveolae number and Cav-1 expression, and determined the effects of caveolae disruption on ET-1, cyclopiazonic acid (CPA) and 1-Oleoyl-2-acetyl-glycerol (OAG)-induced contraction in PAs and Ca(2+) influx in PASMCs of chronic hypoxia (CH)- and monocrotaline (MCT)-induced PH rats. We found that the number of caveolae, and the Cav-1 mRNA and protein levels were increased significantly in PASMCs in both PH models. Disruption of caveolae by cholesterol depletion with methyl-β-cyclodextrin (MβCD) significantly inhibited the contractile response to ET-1, CPA and OAG in PAs of control rats. ET-1, SOCE and ROCE-mediated contractile responses were enhanced, and their susceptibility to MβCD suppression was potentiated in the two PH models. MβCD-induced inhibition was reversed by cholesterol repletion. Introduction of Cav-1 scaffolding domain peptide to mimic Cav-1 upregulation caused significant increase in CPA- and OAG-induced Ca(2+) entry in PASMCs of control, CH and MCT-treated groups. Our results suggest that the increase in caveolae and Cav-1 expression in PH contributes to the enhanced agonist-induced contraction of PA via modulation of SOCE and ROCE; and targeting caveolae/Cav-1 in PASMCs may provide a novel therapeutic strategy for the treatment of PH. PMID:27311393

  18. Protective Effect of Ginsenoside Rg1 on Bleomycin-Induced Pulmonary Fibrosis in Rats: Involvement of Caveolin-1 and TGF-β1 Signal Pathway.

    PubMed

    Zhan, Heqin; Huang, Feng; Ma, Wenzhuo; Zhao, Zhenghang; Zhang, Haifang; Zhang, Chong

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and high mortality rate. Panax Notoginseng Saponins (PNS), extracted from Panax Notoginseng as a traditional Asian medicine, displayed a significant anti-fibrosis effect in liver and lung. However, whether Ginsenoside Rg1 (Rg1), an important and active ingredient of PNS, exerts anti-fibrotic activity on IPF still remain unclear. In this study, we investigated the effect of Rg1 on bleomycin-induced pulmonary fibrosis in rats. Bleomycin (5 mg/kg body weight) was intratracheally administrated to male rats. Rg1 (18, 36 and 72 mg/kg) was orally administered on the next day after bleomycin. Lungs were harvested at day 7 and 28 for the further experiments. Histological analysis revealed that bleomycin successfully induced pulmonary fibrosis, and that Rg1 restored the histological alteration of bleomycin-induced pulmonary fibrosis (PF), significantly decreased lung coefficient, scores of alveolitis, scores of PF as well as contents of alpha smooth muscle actin (α-SMA) and hydroxyproline (Hyp) in a dose-dependent manner in PF rats. Moreover, Rg1 increased the expression levels of Caveolin-1 (Cav-1) mRNA and protein, lowered the expression of transforming growth factor-β1 (TGF-β1) mRNA and protein in the lung tissues of PF rats. These data suggest that Rg1 exhibits protective effect against bleomycin-induced PF in rats, which is potentially associated with the down-regulation of TGF-β1 and up-regulation of Cav-1. PMID:27476938

  19. Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

    PubMed Central

    2014-01-01

    Background Thyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells. Methods Microarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo. Results Using microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model. Conclusions Our findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in

  20. Ubiquitin-specific protease 11 functions as a tumor suppressor by modulating Mgl-1 protein to regulate cancer cell growth

    PubMed Central

    Lim, Key-Hwan; Suresh, Bharathi; Park, Jung-Hyun; Kim, Young-Soo; Ramakrishna, Suresh; Baek, Kwang-Hyun

    2016-01-01

    The Lethal giant larvae (Lgl) gene encodes a cortical cytoskeleton protein, Lgl, and is involved in maintaining cell polarity and epithelial integrity. Previously, we observed that Mgl-1, a mammalian homologue of the Drosophila tumor suppressor protein Lgl, is subjected to degradation via ubiquitin-proteasome pathway, and scaffolding protein RanBPM prevents the turnover of the Mgl-1 protein. Consequently, overexpression of RanBPM enhances Mgl-1-mediated cell proliferation and migration. Here, we analyzed the ability of ubiquitin-specific protease 11 (USP11) as a novel regulator of Mgl-1 and it requires RanBPM to regulate proteasomal degradation of Mgl-1. USP11 showed deubiquitinating activity and stabilized Mgl-1 protein. However, USP11-mediated Mgl-1 stabilization was inhibited in RanBPM-knockdown cells. Furthermore, in the cancer cell migration, the regulation of Mgl-1 by USP11 required RanBPM expression. In addition, an in vivo study revealed that depletion of USP11 leads to tumor formation. Taken together, the results indicated that USP11 functions as a tumor suppressor through the regulation of Mgl-1 protein degradation via RanBPM. PMID:26919101

  1. SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development.

    PubMed

    Bear, J E; Rawls, J F; Saxe, C L

    1998-09-01

    G protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal that is transduced via a family of G protein-coupled receptors, the cARs. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests before tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein was discovered. Loss of this protein, which we call SCAR (suppressor of cAR), restores tip formation and most later development to cAR2(-) strains, and causes a multiple-tip phenotype in a cAR2(+) strain as well as leading to the production of extremely small cells in suspension culture. SCAR-cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologues of SCAR have also been identified in humans, mouse, Caenorhabditis elegans, and Drosophila. These data suggest that SCAR may be a conserved negative regulator of G protein-coupled signaling, and that it plays an important role in regulating the actin cytoskeleton.

  2. HMG-CoA Reductase Inhibitor Improves Endothelial Dysfunction in Spontaneous Hypertensive Rats Via Down-regulation of Caveolin-1 and Activation of Endothelial Nitric Oxide Synthase

    PubMed Central

    Suh, Jung-Won; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10-9-10-4 M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status. PMID:20052342

  3. Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann-Pick type C1.

    PubMed

    Mundy, Dorothy I; Lopez, Adam M; Posey, Kenneth S; Chuang, Jen-Chieh; Ramirez, Charina M; Scherer, Philipp E; Turley, Stephen D

    2014-07-01

    Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1(-/-)), and subsequently in Cav-1(-/-) mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) (Cav-1(-/-):Npc1(-/-)). In 50-day-old Cav-1(-/-) mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1(+/+) controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1(-/-):Npc1(-/-) mice (0.356±0.022) markedly exceeded that in their Cav-1(+/+):Npc1(+/+) controls (0.137±0.009), as well as in their Cav-1(-/-):Npc1(+/+) (0.191±0.013) and Cav-1(+/+):Npc1(-/-) (0.213±0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74±0.17, 0.71±0.05, 0.96±0.05 and 3.12±0.43, respectively, with the extra cholesterol in the Cav-1(-/-):Npc1(-/-) and Cav-1(+/+):Npc1(-/-) mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1(-/-):Npc1(-/-) mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.

  4. Caveolae and caveolin-1 are implicated in 1alpha,25(OH)2-vitamin D3-dependent modulation of Src, MAPK cascades and VDR localization in skeletal muscle cells.

    PubMed

    Buitrago, Claudia; Boland, Ricardo

    2010-07-01

    We previously reported that 1alpha,25(OH)2D3 induces non-transcriptional rapid responses through activation of MAPKs in C2C12 skeletal muscle cells. However, there is little information on the molecular mechanism underlying the initiation of 1alpha,25(OH)2D3 signaling through this pathway. Plasma membrane components have been involved in some non-genomic effects. In this work, we investigated the role of caveolae and caveolin-1 (cav-1) in 1alpha,25(OH)2D3-stimulation of c-Src and MAPKs. When proliferating cells were pretreated with methyl beta cyclodextrin (MbetaCD), a caveolae disrupting agent, under conditions in which cell morphology is not affected and no signs of apoptosis are observed, 1alpha,25(OH)2D3-dependent activation of ERK1/2, p38 MAPK and c-Src was suppressed. Similar results were obtained by siRNA technology whereby silencing of cav-1 expression abolished activation of c-Src and MAPKs induced by the hormone. By confocal immunocytochemistry it was observed that cav-1 colocalizes with c-Src in the periplasma membrane zone at basal conditions. Hormone treatment disrupted the colocalization of these proteins and redistributed them into cytoplasm and nucleus. Co-immunoprecipitation assays corroborated these observations. Changes in VDR localization after 1alpha,25(OH)2D3 exposure were also investigated. Confocal microscopy images showed that the hormone induces VDR translocation to the plasma membrane, and this effect is abolished by MbetaCD. Altogether, these data suggest that caveolae is involved upstream in c-Src-MAPKs activation by 1alpha,25(OH)2D3 and that VDR and cav-1 participate in the rapid signaling elicited by the hormone.

  5. Mild and severe cereal yellow dwarf viruses differ in silencing suppressor efficiency of the P0 protein.

    PubMed

    Almasi, Reza; Miller, W Allen; Ziegler-Graff, Véronique

    2015-10-01

    Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities.

  6. PRMT4-Mediated Arginine Methylation Negatively Regulates Retinoblastoma Tumor Suppressor Protein and Promotes E2F-1 Dissociation

    PubMed Central

    Kim, Kevin Y.; Wang, Don-Hong; Campbell, Mel; Huerta, Steve B.; Shevchenko, Bogdan; Izumiya, Chie

    2014-01-01

    The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb Cterm) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb Cterm phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4–E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1. PMID:25348716

  7. The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

    PubMed

    Li, Chien-Feng; Wu, Wen-Jeng; Wu, Wen-Ren; Liao, Yu-Jing; Chen, Lih-Ren; Huang, Chun-Nung; Li, Ching-Chia; Li, Wei-Ming; Huang, Hsuan-Ying; Chen, Yi-Ling; Liang, Shih-Shin; Chow, Nan-Haw; Shiue, Yow-Ling

    2015-04-20

    In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

  8. Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded

    PubMed Central

    Yu, Jianzhong; Zheng, Yonggang; Dong, Jixin; Klusza, Stephen; Deng, Wu-Min; Pan, Duojia

    2010-01-01

    Summary The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra (Kbr) as another upstream component of the Hippo signaling pathway. We show that Kbr functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function, and implicate Kbr as a potential tumor suppressor with relevance to neurofibromatosis. PMID:20159598

  9. Src-mediated caveolin-1 phosphorylation regulates intestinal epithelial restitution by altering Ca2+ influx after wounding

    PubMed Central

    Rathor, Navneeta; Zhuang, Ran; Wang, Jian-Ying; Donahue, James M.; Turner, Douglas J.

    2014-01-01

    Early mucosal restitution occurs as a consequence of intestinal epithelial cell (IEC) migration to reseal superficial wounds, but its exact mechanism remains largely unknown. Caveolin-1 (Cav1), a major component associated with caveolar lipid rafts in the plasma membrane, is implicated in many aspects of cellular functions. This study determined if c-Src kinase (Src)-induced Cav1 phosphorylation promotes intestinal epithelial restitution after wounding by activating Cav1-mediated Ca2+ signaling. Src directly interacted with Cav1, formed Cav1-Src complexes, and phosphorylated Cav1 in IECs. Inhibition of Src activity by its chemical inhibitor PP2 or suppression of the functional caveolin scaffolding domain by caveolin-scaffolding domain peptides prevented Cav1-Src interaction, reduced Cav1 phosphorylation, decreased Ca2+ influx, and inhibited cell migration after wounding. Disruption of caveolar lipid raft microdomains by methyl-β-cyclodextrin reduced Cav1-mediated Ca2+ influx and repressed epithelial restitution. Moreover, Src silencing prevented subcellular redistribution of phosphorylated Cav1 in migrating IECs. These results indicate that Src-induced Cav1 phosphorylation stimulates epithelial restitution by increasing Cav1-mediated Ca2+ signaling after wounding, thus contributing to the maintenance of gut mucosal integrity under various pathological conditions. PMID:24557763

  10. Tyrosine-Phosphorylated Caveolin-1 Blocks Bacterial Uptake by Inducing Vav2-RhoA-Mediated Cytoskeletal Rearrangements

    PubMed Central

    Kaushansky, Alexis; Pompaiah, Malvika; Thorn, Hans; Brinkmann, Volker; MacBeath, Gavin; Meyer, Thomas F.

    2010-01-01

    Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P+GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function. PMID:20808760

  11. Elicitation of hypersensitive responses in Nicotiana glutinosa by the suppressor of RNA silencing protein P0 from poleroviruses.

    PubMed

    Wang, Ken-Der; Empleo, Roman; Nguyen, Tan Tri V; Moffett, Peter; Sacco, Melanie Ann

    2015-06-01

    Plant disease resistance (R) proteins that confer resistance to viruses recognize viral gene products with diverse functions, including viral suppressors of RNA silencing (VSRs). The P0 protein from poleroviruses is a VSR that targets the ARGONAUTE1 (AGO1) protein for degradation, thereby disrupting RNA silencing and antiviral defences. Here, we report resistance against poleroviruses in Nicotiana glutinosa directed against Turnip yellows virus (TuYV) and Potato leafroll virus (PLRV). The P0 proteins from TuYV (P0(T) (u) ), PLRV (P0(PL) ) and Cucurbit aphid-borne yellows virus (P0(CA) ) were found to elicit a hypersensitive response (HR) in N. glutinosa accession TW59, whereas other accessions recognized P0(PL) only. Genetic analysis showed that recognition of P0(T) (u) by a resistance gene designated RPO1 (Resistance to POleroviruses 1) is inherited as a dominant allele. Expression of P0 from a Potato virus X (PVX) expression vector transferred recognition to the recombinant virus on plants expressing RPO1, supporting P0 as the unique Polerovirus factor eliciting resistance. The induction of HR required a functional P0 protein, as P0(T) (u) mutants with substitutions in the F-box motif that abolished VSR activity were unable to elicit HR. We surmised that the broad P0 recognition seen in TW59 and the requirement for the F-box protein motif could indicate detection of P0-induced AGO1 degradation and disruption of RNA silencing; however, other viral silencing suppressors, including the PVX P25 that also causes AGO1 degradation, failed to elicit HR in N. glutinosa. Investigation of P0 elicitation of RPO1 could provide insight into P0 activities within the cell that trigger resistance.

  12. The G-protein Alpha Subunit Gsα Is A Tumor Suppressor In Sonic Hedgehog-driven Medulloblastoma

    PubMed Central

    He, Xuelian; Zhang, Liguo; Chen, Ying; Remke, Marc; Shih, David; Lu, Fanghui; Wang, Haibo; Deng, Yaqi; Yu, Yang; Xia, Yong; Wu, Xiaochong; Ramaswamy, Vijay; Hu, Tom; Wang, Fan; Zhou, Wenhao; Burns, Dennis K.; Kim, Se Hoon; Kool, Marcel; Pfister, Stefan M.; Weinstein, Lee S.; Pomeroy, Scott L.; Gilbertson, Richard J.; Rubin, Joshua B.; Hou, Yiping; Wechsler-Reya, Robert; Taylor, Michael D.; Lu, Q. Richard

    2014-01-01

    Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G-protein Gsα, as a potent tumor suppressor gene that defines a subset of aggressive Sonic Hedgehog (Shh)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically-distinct progenitors is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gsα is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh-signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation of a Gsα effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas mutants. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gsα that acts as a molecular link across Shh-group medulloblastomas of disparate cellular and anatomical origins, illuminating G-protein modulation as a potential therapeutic avenue. PMID:25150496

  13. Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster

    SciTech Connect

    Holdridge, C.; Dorsett, D. )

    1991-04-01

    The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. The authors found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. They propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

  14. Nasopharyngeal carcinomas frequently lack the p16/MTS1 tumor suppressor protein but consistently express the retinoblastoma gene product.

    PubMed Central

    Gulley, M. L.; Nicholls, J. M.; Schneider, B. G.; Amin, M. B.; Ro, J. Y.; Geradts, J.

    1998-01-01

    The p16/MTS1 gene is altered by deletion, mutation, or hypermethylation in a wide variety of human cancers. As a result of deficient p16 protein, these cancers lack a critical mechanism for halting G1/S cell cycle progression. In the current study, 59 cases of nasopharyngeal carcinoma were evaluated for expression of the p16 tumor suppressor protein by immunohistochemical analysis of paraffin-embedded tissue. There was no detectable p16 in 38/59 cases (64%), which implies a very high rate of p16 inactivation in this type of cancer. On the other hand, the retinoblastoma gene product, which also regulates the G1 to S phase transition of the cell cycle, was consistently expressed in nasopharyngeal carcinomas by immunohistochemical analysis. These results implicate p16 inactivation but not Rb alteration in the stepwise progression of nasopharyngeal carcinogenesis. Images Figure 1 Figure 2 PMID:9546345

  15. Cop1 constitutively regulates c-Jun protein stability and functions as a tumor suppressor in mice

    PubMed Central

    Migliorini, Domenico; Bogaerts, Sven; Defever, Dieter; Vyas, Rajesh; Denecker, Geertrui; Radaelli, Enrico; Zwolinska, Aleksandra; Depaepe, Vanessa; Hochepied, Tino; Skarnes, William C.; Marine, Jean-Christophe

    2011-01-01

    Biochemical studies have suggested conflicting roles for the E3 ubiquitin ligase constitutive photomorphogenesis protein 1 (Cop1; also known as Rfwd2) in tumorigenesis, providing evidence for both the oncoprotein c-Jun and the tumor suppressor p53 as its targets. Here we present what we believe to be the first in vivo investigation of the role of Cop1 in cancer etiology. Using an innovative genetic approach to generate an allelic series of Cop1, we found that Cop1 hypomorphic mice spontaneously developed malignancy at a high frequency in the first year of life and were highly susceptible to radiation-induced lymphomagenesis. Further analysis revealed that c-Jun was a key physiological target for Cop1 and that Cop1 constitutively kept c-Jun at low levels in vivo and thereby modulated c-Jun/AP-1 transcriptional activity. Importantly, Cop1 deficiency stimulated cell proliferation in a c-Jun–dependent manner. Focal deletions of COP1 were observed at significant frequency across several cancer types, and COP1 loss was determined to be one of the mechanisms leading to c-Jun upregulation in human cancer. We therefore conclude that Cop1 is a tumor suppressor that functions, at least in part, by antagonizing c-Jun oncogenic activity. In the absence of evidence for a genetic interaction between Cop1 and p53, our data strongly argue against the use of Cop1-inhibitory drugs for cancer therapy. PMID:21403399

  16. Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors.

    PubMed

    Martínez-Turiño, Sandra; Hernández, Carmen

    2009-02-01

    Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

  17. Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann-Pick type C1

    PubMed Central

    Mundy, Dorothy I.; Lopez, Adam M.; Posey, Kenneth S.; Chuang, Jen-Chieh; Ramirez, Charina M.; Scherer, Philipp E.; Turley, Stephen D.

    2014-01-01

    Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1−/−), and subsequently in Cav-1−/− mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) was also absent (Cav-1−/−:Npc1−/−). In 50-day-old Cav-1−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1−/−:Npc1−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1+/+:Npc1+/+ controls (0.137 ± 0.009), as well as in their Cav-1−/−:Npc1+/+ (0.191 ± 0.013) and Cav-1+/+:Npc1−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol content (mg/organ) in mice of these genotypes was 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1−/−:Npc1−/− and Cav-1+/+:Npc1−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1−/−:Npc1−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted. PMID:24747682

  18. Role of caveolin 1 in AT1a receptor-mediated uptake of angiotensin II in the proximal tubule of the kidney

    PubMed Central

    Li, Xiao C.; Gu, Victor; Miguel-Qin, Elise

    2014-01-01

    Caveolin 1 (CAV-1) functions not only as a constitutive scaffolding protein of caveolae but also as a vesicular transporter and signaling regulator. In the present study, we tested the hypothesis that CAV-1 knockout (CAV-1 KO) inhibits ANG II type 1 [AT1 (AT1a)] receptor-mediated uptake of ANG II in the proximal tubule and attenuates blood pressure responses in ANG II-induced hypertension. To determine the role of CAV-1 in mediating the uptake of FITC-labeled ANG II, wild-type (WT) mouse proximal convoluted tubule cells were transfected with CAV-1 small interfering (si)RNA for 48 h before AT1 receptor-mediated uptake of FITC-labeled ANG II was studied. CAV-1 siRNA knocked down CAV-1 expression by >90% (P < 0.01) and inhibited FITC-labeled ANG II uptake by >50% (P < 0.01). Moreover, CAV-1 siRNA attenuated ANG II-induced activation of MAPK ERK1/2 and Na+/H+ exchanger 3 expression, respectively (P < 0.01). To determine whether CAV-1 regulates ANG II uptake in the proximal tubule, Alexa 488-labeled ANG II was infused into anesthetized WT and CAV-1 KO mice for 60 min (20 ng/min iv). Imaging analysis revealed that Alexa 488-labeled ANG II uptake was decreased by >50% in CAV-1 KO mice (P < 0.01). Furthermore, Val5-ANG II was infused into WT and CAV-1 KO mice for 2 wk (1.5 mg·kg−1·day−1 ip). Basal systolic pressure was higher, whereas blood pressure and renal excretory and signaling responses to ANG II were attenuated, in CAV-1 KO mice (P < 0.01). We concluded that CAV-1 plays an important role in AT1 receptor-mediated uptake of ANG II in the proximal tubule and modulates blood pressure and renal responses to ANG II. PMID:25164083

  19. Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells.

    PubMed

    Kwok, Willson; Lee, Sang Ho; Culberson, Cathy; Korneszczuk, Katarzyna; Clemens, Mark G

    2009-11-01

    Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-beta-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 microg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

  20. Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells.

    PubMed

    Shen, Jiangang; Lee, Waisin; Li, Yue; Lau, Chi Fai; Ng, Kwong Man; Fung, Man Lung; Liu, Ke Jian

    2008-10-01

    Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells. PMID:18717816

  1. Targeting cysteine rich C1 domain of Scaffold protein Kinase Suppressor of Ras (KSR) with anthocyanidins and flavonoids - a binding affinity characterization study.

    PubMed

    Karthik, Dhananjayan; Majumder, Pulak; Palanisamy, Sivanandy; Khairunnisa, Kalathil; Venugopal, Varsha

    2014-01-01

    Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this mechanism can have a high influence in inhibiting the downstream signaling of the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of the studies targeted to prevent the binding of Raf, MEK binding on kinase suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding site with small ligand molecules like flavones and anthocyanidins and analyzed through insilico docking studies. The binding energy evaluation shows the importance of hydroxyl groups at various positions on the flavone and anthocyanidin nucleus. Over all binding interaction shows these ligands occupied the potential sites of cysteine rich C1 domain of scaffold protein KSR. PMID:25352726

  2. The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat.

    PubMed

    Schnettler, Esther; de Vries, Walter; Hemmes, Hans; Haasnoot, Joost; Kormelink, Richard; Goldbach, Rob; Berkhout, Ben

    2009-03-01

    The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double-stranded RNA (dsRNA) as a common inducer molecule. The non-structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules. Here, we show that this plant viral RSS lacks IFN antagonistic activity, yet it is able to substitute the RSS function of the Tat protein of human immunodeficiency virus type 1. An NS3 mutant that is deficient in RNA binding and its associated RSS activity is inactive in this complementation assay. This cross-kingdom suppression of RNAi in mammalian cells by a plant viral RSS indicates the significance of the antiviral RNAi response in mammalian cells and the usefulness of well-defined RSS proteins. PMID:19218918

  3. The negative regulator of Gli, Suppressor of fused (Sufu), interacts with SAP18, Galectin3 and other nuclear proteins.

    PubMed Central

    Paces-Fessy, Mélanie; Boucher, Dominique; Petit, Emile; Paute-Briand, Sandrine; Blanchet-Tournier, Marie-Françoise

    2004-01-01

    Sufu (Suppressor of fused) is a negative regulator of the Hedgehog signal-transduction pathway, interacting directly with the Gli family of transcription factors. However, its function remains poorly understood. In the present study, we determined the expression, tissue distribution and biochemical properties of mSufu (mouse Sufu) protein. We identified several mSufu variants of which some were phosphorylated. A yeast two-hybrid screen with mSufu as bait allowed us to identify several nuclear proteins as potential partners of mSufu. Most of these partners, such as SAP18 (Sin3-associated polypeptide 18), pCIP (p300/CBP-cointegrator protein) and PIAS1 (protein inhibitor of activated signal transduction and activators of transcription 1), are involved in either repression or activation of transcription and two of them, Galectin3 and hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), have a nuclear function in pre-mRNA splicing. We confirmed the mSufu-SAP18 and mSufu-Galectin3 interactions by independent biochemical assays. Using a cell transfection assay, we also demonstrated that mSufu protein (484 amino acids) is predominantly cytoplasmic but becomes mostly nuclear when a putative nuclear export signal is mutated or after treatment of the cells with leptomycin B. Moreover, mSufu is translocated to the nucleus when co-expressed with SAP18, which is normally found in this compartment. In contrast, Galectin3 is translocated to the cytoplasm when it is co-expressed with mSufu. Our findings indicate that mSufu is a shuttle protein that appears to be extremely versatile in its ability to bind different proteins in both the cytoplasm and nucleus. PMID:14611647

  4. Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

    PubMed

    Lee, So-Youn; Yi, Jin-Kyu; Yun, Hyung-Mun; Bae, Cheol-Hyeon; Cho, Eui-Sic; Lee, Kook-Sun; Kim, Eun-Cheol

    2016-05-01

    It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-β-cyclodextrin (MβCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MβCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MβCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3β, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be

  5. Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers.

    PubMed

    Witkiewicz, Agnieszka K; Dasgupta, Abhijit; Sammons, Sara; Er, Ozlem; Potoczek, Magdalena B; Guiles, Fran; Sotgia, Federica; Brody, Jonathan R; Mitchell, Edith P; Lisanti, Michael P

    2010-07-15

    Here, we investigated the possible predictive value of stromal caveolin-1 (Cav-1) as a candidate biomarker for clinical outcome in triple negative (TN) breast cancer patients. A cohort of 85 TN breast cancer patients was available, with the necessary annotation and nearly 12 years of follow-up data. Our primary outcome of interest in this study was overall survival. Interestingly, TN patients with high-levels of stromal Cav-1 had a good clinical outcome, with >50% of the patients remaining alive during the follow-up period. In contrast, the median survival for TN patients with moderate stromal Cav-1 staining was 33.5 months. Similarly, the median survival for TN patients with absent stromal Cav-1 staining was 25.7 months. A comparison of 5-year survival rates yields a similar pattern. TN patients with high stromal Cav-1 had a good 5-year survival rate, with 75.5% of the patients remaining alive. In contrast, TN patients with moderate or absent stromal Cav-1 levels had progressively worse 5-year survival rates, with 40 and 9.4% of the patients remaining alive. In contrast, in a parallel analysis, the levels of tumor epithelial Cav-1 had no prognostic significance. As such, the prognostic value of Cav-1 immunostaining in TN breast cancer patients is compartment-specific, and selective for an absence of Cav-1 staining in the stromal fibroblast compartment. A recursive-partitioning algorithm was used to assess which factors are most predictive of overall survival in TN breast cancer patients. In this analysis, we included tumor size, histologic grade, whether the patient received surgery, radiotherapy or chemotherapy, CK5/6, EGFR, p53 and Ki67 status, as well as the stromal Cav-1 score. This analysis indicated that stromal loss of Cav-1 expression was the most important prognostic factor for overall survival in TN breast cancer. Virtually identical results were obtained with CK5/6 (+) and/or EGFR (+) TN breast cancer cases, demonstrating that a loss of stromal Cav-1 is

  6. The G protein Gαs acts as a tumor suppressor in sonic hedgehog signaling-driven tumorigenesis.

    PubMed

    Rao, Rohit; Salloum, Ralph; Xin, Mei; Lu, Q Richard

    2016-05-18

    G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gαs signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gαs can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gαs suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gαs, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gαs activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gαs-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gαs in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression. PMID:27052725

  7. Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies.

    PubMed

    Routila, Johannes; Mäkelä, Juho-Antti; Luukkaa, Heikki; Leivo, Ilmo; Irjala, Heikki; Westermarck, Jukka; Mäkitie, Antti; Ventelä, Sami

    2016-01-01

    The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

  8. Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains.

    PubMed Central

    Carruth, L M; Hardwick, J M; Morse, B A; Clements, J E

    1994-01-01

    Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat. Images PMID:8083955

  9. Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Scotcher, Jenna; Clarke, David J.; Weidt, Stefan K.; Mackay, C. Logan; Hupp, Ted R.; Sadler, Peter J.; Langridge-Smith, Pat R. R.

    2011-05-01

    The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

  10. Overexpression of tumor suppressor protein OSCP1/NOR1 induces ER stress and apoptosis during development of Drosophila melanogaster

    PubMed Central

    Huu, Nguyen Tho; Yoshida, Hideki; Yamaguchi, Masamitsu

    2015-01-01

    OSCP1/NOR1 (organic solute carrier partner 1/oxidored-nitro domain-containing protein 1) is known as a transporter of various organic solutes into cells and also is reported to act as a tumor suppressor protein. Although overexpression of OSCP1 has been shown to play multiple roles in mammalian cell lines, its biological significance in living organisms is not fully understood. To explore the effects of OSCP1/NOR1 on development, we performed genetic studies in flies featuring overexpression of its Drosophila orthologue, dOSCP1. Overexpression of dOSCP1 in eye imaginal discs induced a rough eye phenotype in adult flies, likely resulting from a delay in S phase progression and induction of caspase-dependent apoptosis followed by compensatory proliferation. However, it did not appear to be involved in differentiation of R7 photoreceptor cells. We also found that overexpression of dOSCP1 caused endoplasmic reticulum stress in salivary gland cells. These results indicate that overexpression of dOSCP1 exerts effects on various biological processes during Drosophila development. PMID:26175940

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

    PubMed

    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.

  12. The Drosophila suppressor of underreplication protein binds to late-replicating regions of polytene chromosomes.

    PubMed Central

    Makunin, I V; Volkova, E I; Belyaeva, E S; Nabirochkina, E N; Pirrotta, V; Zhimulev, I F

    2002-01-01

    In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin. PMID:11901119

  13. Conserved Molecular Underpinnings and Characterization of a Role for Caveolin-1 in the Tumor Microenvironment of Mature T-Cell Lymphomas

    PubMed Central

    Herek, Tyler A.; Shew, Timothy D.; Spurgin, Heather N.; Cutucache, Christine E.

    2015-01-01

    Neoplasms of extra-thymic T-cell origin represent a rare and difficult population characterized by poor clinical outcome, aggressive presentation, and poorly defined molecular characteristics. Much work has been done to gain greater insights into distinguishing features among malignant subtypes, but there also exists a need to identify unifying characteristics to assist in rapid diagnosis and subsequent potential treatment. Herein, we investigated gene expression data of five different mature T-cell lymphoma subtypes (n = 187) and found 21 genes to be up- and down-regulated across all malignancies in comparison to healthy CD4+ and CD8+ T-cell controls (n = 52). From these results, we sought to characterize a role for caveolin-1 (CAV1), a gene with previous description in the progression of both solid and hematological tumors. Caveolin-1 was upregulated, albeit with a heterogeneous nature, across all mature T-cell lymphoma subtypes, a finding confirmed using immunohistochemical staining on an independent sampling of mature T-cell lymphoma biopsies (n = 65 cases). Further, stratifying malignant samples in accordance with high and low CAV1 expression revealed that higher expression of CAV1 in mature T-cell lymphomas is analogous with an enhanced inflammatory and invasive gene expression profile. Taken together, these results demonstrate a role for CAV1 in the tumor microenvironment of mature T-cell malignancies and point toward potential prognostic implications. PMID:26566034

  14. Nano-Mg(OH)2-induced proliferation inhibition and dysfunction of human umbilical vein vascular endothelial cells through caveolin-1-mediated endocytosis.

    PubMed

    Meng, Ning; Han, Lei; Pan, XiaoHong; Su, Le; Jiang, Zheng; Lin, Zhang; Zhao, Jing; Zhang, ShangLi; Zhang, Yun; Zhao, BaoXiang; Miao, JunYing

    2015-02-01

    Nano-Mg(OH)2 is efficiently used in pollutant adsorption and removal due to its high adsorption capability, low-cost, and recyclability. A recent research from our group showed that Mg(OH)2 nanoflakes are not evidently internalized by cancer cells and are not cytotoxic. But the biocompatibility and potential toxicity of nano-Mg(OH)2 in a normal biological system are largely unclear. Nanoparticles could affect the function of endothelial cells, and endothelial dysfunction represents an early sign of lesion within the vasculature. Here, we applied the human umbilical vein vascular endothelial cells (HUVECs) as an in vitro model of the endothelium to study the cytotoxicity of nano-Mg(OH)2. Our results showed that nano-Mg(OH)2 at 200 μg/ml impaired proliferation and induced dysfunction of HUVECs, but did not result in cell necrosis and apoptosis. Transmission electron microscopy images and immunofluorescence results showed that the nano-Mg(OH)2 could enter HUVECs through caveolin-1-mediated endocytosis. Nano-Mg(OH)2 at high concentrations decreased the level of caveolin-1 and increased the activity of endothelial nitric oxide synthase (eNOS), thus leading to the production of excess nitric oxide (NO). In this work, we provide the cell damage concentrations of nano-Mg(OH)2 nanoparticles, and we propose a mechanism of injury induced by nano-Mg(OH)2 in HUVECs.

  15. An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

    PubMed

    Bromley, Dennis; Bauer, Matthias R; Fersht, Alan R; Daggett, Valerie

    2016-09-01

    The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant. PMID:27503952

  16. An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

    PubMed

    Bromley, Dennis; Bauer, Matthias R; Fersht, Alan R; Daggett, Valerie

    2016-09-01

    The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

  17. C2-streptavidin mediates the delivery of biotin-conjugated tumor suppressor protein p53 into tumor cells.

    PubMed

    Fahrer, Jörg; Schweitzer, Brigitte; Fiedler, Katja; Langer, Torben; Gierschik, Peter; Barth, Holger

    2013-04-17

    We have previously generated a recombinant C2-streptavidin fusion protein for the delivery of biotin-labeled molecules of low molecular weight into the cytosol of mammalian cells. A nontoxic moiety of Clostridium botulinum C2 toxin mediates the cellular uptake, whereas the streptavidin unit serves as a binding platform for biotin-labeled cargo molecules. In the present study, we used the C2-streptavidin transporter to introduce biotin-conjugated p53 protein into various mammalian cell lines. The p53 tumor suppressor protein is inactivated in many human cancers by multiple mechanisms and therefore the restoration of its activity in tumor cells is of great therapeutic interest. Recombinant p53 was expressed in insect cells and biotin-labeled. Biotin-p53 retained its specific high-affinity DNA-binding as revealed by gel-shift analysis. Successful conjugation of biotin-p53 to the C2-streptavidin transporter was monitored by an overlay blot technique and confirmed by real-time surface plasmon resonance, providing a KD-value in the low nM range. C2-streptavidin significantly enhanced the uptake of biotin-p53 into African Green Monkey (Vero) epithelial cells as shown by flow cytometry. Using cell fractionation, the cytosolic translocation of biotin-p53 was detected in Vero cells as well as in HeLa cervix carcinoma cells. In line with this finding, confocal microscopy displayed cytoplasmic staining of biotin-p53 in HeLa and HL60 leukemia cells. Internalized biotin-p53 partially colocalized with early endosomes, as confirmed by confocal microscopy. In conclusion, our results demonstrate the successful conjugation of biotin-p53 to C2-streptavidin and its subsequent receptor-mediated endocytosis into different human tumor cell lines.

  18. HBP21, a chaperone of heat shock protein 70, functions as a tumor suppressor in hepatocellular carcinoma.

    PubMed

    Jiang, Lingxi; Kwong, Dora Lai-Wan; Li, Yan; Liu, Ming; Yuan, Yun-Fei; Li, Yan; Fu, Li; Guan, Xin-Yuan

    2015-10-01

    Inactivation of tumor suppressor genes, caused by genetic and epigenetic alterations, is one of the key issues in the development and progression of cancer. To identify and characterize cancer related genes in hepatocellular carcinoma (HCC) pathogenesis, transcriptome sequencing has been applied to compare expression profiles between tumor and non-tumor tissues. Among the down-regulated genes, heat shock binding protein 21 (HBP21) was selected for further study. In this study, down-regulation of HBP21 was frequently detected in primary HCCs (87/120, 72.5%), which was significantly associated with advanced clinical stage (P = 0.049), poor differentiation (P = 0.018) and poor prognosis (P = 0.026). Further study found that down-regulation of HBP21 in HCC was mainly caused by allele loss and promoter methylation. Functional study found that HBP21 could inhibit tumor cell growth rate, foci formation and colony formation in soft agar, and tumor formation in nude mice when it was transfected into HCC cells. Molecular study found that HBP21 could promote cell apoptosis, especially under adverse conditions such as heat and chemotherapeutic agent treatment. As a chaperone of heat shock protein 70 (HSP70), HBP21 could inhibit interaction between HSP70 and Bax, increased Bax protein translocation from cytoplasm to mitochondria, and subsequently increased the release of cytochrome c into cytoplasm, and finally induced apoptosis. Clinically, HBP21 could be used as a prognostic biomarker for HCC outcome prediction and might be also as a novel therapeutic agent in HCC treatment.

  19. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    PubMed

    Simeon, Rudo L; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides. PMID:24391867

  20. A Screen for Genetic Suppressor Elements of Hepatitis C Virus Identifies a Supercharged Protein Inhibitor of Viral Replication

    PubMed Central

    Simeon, Rudo L.; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1’s anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides. PMID:24391867

  1. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    PubMed

    Simeon, Rudo L; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

  2. Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide–High Angiotensin II–Induced Cardiovascular Injury

    PubMed Central

    Pojoga, Luminita H.; Yao, Tham M.; Opsasnick, Lauren A.; Siddiqui, Waleed T.; Reslan, Ossama M.; Adler, Gail K.; Williams, Gordon H.

    2015-01-01

    Aldosterone interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors and also endothelial nitric oxide synthase, thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1–replete or –deficient states would alter vascular function in a mouse model of low nitric oxide (NO)–high angiotensin II (AngII)–induced cardiovascular injury. Wild-type (WT) and cav-1 knockout mice (cav-1−/−) consuming a high salt diet (4% NaCl) received Nω-nitro-l-arginine methyl ester (L-NAME) (0.1–0.2 mg/ml in drinking water at days 1–11) plus AngII (0.7–2.8 mg/kg per day via an osmotic minipump at days 8–11) ± MR antagonist eplerenone (EPL) 100 mg/kg per day in food. In both genotypes, blood pressure increased with L-NAME + AngII. EPL minimally changed blood pressure, although its dose was sufficient to block MR and reverse cardiac expression of the injury markers cluster of differentiation 68 and plasminogen activator inhibitor-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine and KCl contraction was enhanced with EPL in L-NAME+AngII treated WT mice, but not cav-1−/− mice. AngII-induced contraction was not different, and angiotensin type 1 receptor expression was reduced in L-NAME + AngII treated WT and cav-1−/− mice. In WT mice, acetylcholine-induced relaxation was enhanced with L-NAME + AngII treatment and reversed with EPL. Acetylcholine relaxation in cav-1−/− mice was greater than in WT mice, not modified by L-NAME + AngII or EPL, and blocked by ex vivo L-NAME, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or endothelium removal, suggesting the role of NO-cGMP. Cardiac endothelial NO synthase was increased in cav-1−/− versus WT mice, further increased with L-NAME + AngII, and

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

    PubMed

    Hamada, Fumihiko; Bienz, Mariann

    2004-11-01

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

  4. MicroRNA-326 functions as a tumor suppressor in colorectal cancer by targeting the nin one binding protein.

    PubMed

    Wu, Lei; Hui, Hui; Wang, Li-Juan; Wang, Hao; Liu, Qiu-Fang; Han, Su-Xia

    2015-05-01

    Accumulating evidence has demonstrated that microRNAs (miRNAs) are involved in multiple processes in cancer development and progression. miR-326 has been identified as a tumor suppressor miRNA in several types of human cancer. However, the specific function of miR-326 and its target the nin one binding protein (NOB1) in colorectal carcinoma (CRC) remains unclear. In the present study, we found that miR-326 inhibited cell proliferation, migration and invasion, and induced cell apoptosis and cell cycle arrest of CRC cells by directly targeting NOB1. Furthermore, the upregulation of miR-326 in CRC cells was revealed to be associated with a feedback loop involving downregulation of the NOB1, which mimics the phenotype induced by miR-326. Importantly, we found that the CRC patients with high expression of miR-326 or low expression of NOB1 tend to obtain a better prognosis. Thus, for the first time, we provide convincing evidence that downregulation of miR-326 inhibited tumor proliferation and tumor metastasis by directly targeting NOB1 in CRC. NOB1 and miR-326 could be potential therapeutic targets for CRC.

  5. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  6. Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein.

    PubMed

    Cascales, Eric; Atmakuri, Krishnamohan; Liu, Zhenying; Binns, Andrew N; Christie, Peter J

    2005-10-01

    Agrobacterium tumefaciens uses a type IV secretion (T4S) system composed of VirB proteins and VirD4 to deliver oncogenic DNA (T-DNA) and protein substrates to susceptible plant cells during the course of infection. Here, by use of the Transfer DNA ImmunoPrecipitation (TrIP) assay, we present evidence that the mobilizable plasmid RSF1010 (IncQ) follows the same translocation pathway through the VirB/D4 secretion channel as described previously for the T-DNA. The RSF1010 transfer intermediate and the Osa protein of plasmid pSa (IncW), related in sequence to the FiwA fertility inhibition factor of plasmid RP1 (IncPalpha), render A. tumefaciens host cells nearly avirulent. By use of a semi-quantitative TrIP assay, we show that both of these 'oncogenic suppressor factors' inhibit binding of T-DNA to the VirD4 substrate receptor. Both factors also inhibit binding of the VirE2 protein substrate to VirD4, as shown by coimmunoprecipitation and bimolecular fluorescence complementation assays. Osa fused to the green fluorescent protein (GFP) also blocks T-DNA and VirE2 binding to VirD4, and Osa-GFP colocalizes with VirD4 at A. tumefaciens cell poles. RSF1010 and Osa interfere specifically with VirD4 receptor function and not with VirB channel activity, as shown by (i) TrIP and (ii) a genetic screen for effects of the oncogenic suppressors on pCloDF13 translocation through a chimeric secretion channel composed of the pCloDF13-encoded MobB receptor and VirB channel subunits. Our findings establish that a competing plasmid substrate and a plasmid fertility inhibition factor act on a common target, the T4S receptor, to inhibit docking of DNA and protein substrates to the translocation apparatus. PMID:16194240

  7. Tissue-specific autoregulation of Drosophila suppressor of forked by alternative poly(A) site utilization leads to accumulation of the suppressor of forked protein in mitotically active cells.

    PubMed

    Juge, F; Audibert, A; Benoit, B; Simonelig, M

    2000-11-01

    The Suppressor of forked protein is the Drosophila homolog of the 77K subunit of human cleavage stimulation factor, a complex required for the first step of the mRNA 3'-end-processing reaction. We have shown previously that wild-type su(f) function is required for the accumulation of a truncated su(f) transcript polyadenylated in intron 4 of the gene. This led us to propose a model in which the Su(f) protein would negatively regulate its own accumulation by stimulating 3'-end formation of this truncated su(f) RNA. In this article, we demonstrate this model and show that su(f) autoregulation is tissue specific. The Su(f) protein accumulates at a high level in dividing tissues, but not in nondividing tissues. We show that this distribution of the Su(f) protein results from stimulation by Su(f) of the tissue-specific utilization of the su(f) intronic poly(A) site, leading to the accumulation of the truncated su(f) transcript in nondividing tissues. Utilization of this intronic poly(A) site is affected in a su(f) mutant and restored in the mutant with a transgene encoding wild-type Su(f) protein. These data provide an in vivo example of cell-type-specific regulation of a protein level by poly(A) site choice, and confirm the role of Su(f) in regulation of poly(A) site utilization.

  8. Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML)

    PubMed Central

    Milutinovic, Snezana; Heynen-Genel, Susanne; Chao, Elizabeth; Dewing, Antimone; Solano, Ricardo; Milan, Loribelle; Barron, Nikki; He, Min; Diaz, Paul W.; Matsuzawa, Shu-ichi; Reed, John C.; Hassig, Christian A.

    2016-01-01

    Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli. PMID:27031987

  9. Activation of protein phosphatase 2A tumor suppressor as potential treatment of pancreatic cancer

    PubMed Central

    Chien, Wenwen; Sun, Qiao-Yang; Lee, Kian Leong; Ding, Ling-Wen; Wuensche, Peer; Torres-Fernandez, Lucia A.; Tan, Siew Zhuan; Tokatly, Itay; Zaiden, Norazean; Poellinger, Lorenz; Mori, Seiichi; Yang, Henry; Tyner, Jeffrey W.; Koeffler, H. Phillip

    2015-01-01

    We utilized three tiers of screening to identify novel therapeutic agents for pancreatic cancers. First, we analyzed 14 pancreatic cancer cell lines against a panel of 66 small-molecule kinase inhibitors and dasatinib was the most potent. Second, we performed RNA expression analysis on 3 dasatinib-resistant and 3 dasatinib–sensitive pancreatic cancer cell lines to profile their gene expression. Third, gene profiling data was integrated with the connectivity map database to search for potential drugs. Thioridazine was one of the top ranking small molecules with highly negative enrichment. Thioridazine and its family members of phenothiazine including penfludidol caused pancreatic cancer cell death and affected protein expression levels of molecules involved in cell cycle regulation, apoptosis, and multiple kinase activities. This family of drugs causes activation of protein phosphatase 2 (PP2A). The drug FTY-720 (activator of PP2A) induced apoptosis of pancreatic cancer cells. Silencing catalytic unit of PP2A rendered pancreatic cancer cells resistant to penfluridol. Our observations suggest potential therapeutic use of penfluridol or similar agent associated with activation of PP2A in pancreatic cancers. PMID:25637283

  10. Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML).

    PubMed

    Milutinovic, Snezana; Heynen-Genel, Susanne; Chao, Elizabeth; Dewing, Antimone; Solano, Ricardo; Milan, Loribelle; Barron, Nikki; He, Min; Diaz, Paul W; Matsuzawa, Shu-ichi; Reed, John C; Hassig, Christian A

    2016-01-01

    Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion. CG effects on PML occur at the post-transcriptional level, mechanistically distinct from previously described PML activators and are mediated through signaling events downstream of NKA. Curiously, genomic deletion of PML in human cancer cells failed to abrogate the cytotoxic effects of CGs and other apoptotic stimuli such as ceramide and arsenic trioxide that were previously shown to function through PML in mice. These findings suggest that alternative pathways can compensate for PML loss to mediate apoptosis in response to CGs and other apoptotic stimuli. PMID:27031987

  11. Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein

    PubMed Central

    Payankaulam, Sandhya; Yeung, Kelvin; McNeill, Helen; Henry, R. William; Arnosti, David N.

    2016-01-01

    In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical–basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis. PMID:26971715

  12. Regulation of cell polarity determinants by the Retinoblastoma tumor suppressor protein.

    PubMed

    Payankaulam, Sandhya; Yeung, Kelvin; McNeill, Helen; Henry, R William; Arnosti, David N

    2016-01-01

    In addition to their canonical roles in the cell cycle, RB family proteins regulate numerous developmental pathways, although the mechanisms remain obscure. We found that Drosophila Rbf1 associates with genes encoding components of the highly conserved apical-basal and planar cell polarity pathways, suggesting a possible regulatory role. Here, we show that depletion of Rbf1 in Drosophila tissues is indeed associated with polarity defects in the wing and eye. Key polarity genes aPKC, par6, vang, pk, and fmi are upregulated, and an aPKC mutation suppresses the Rbf1-induced phenotypes. RB control of cell polarity may be an evolutionarily conserved function, with important implications in cancer metastasis. PMID:26971715

  13. New functional and biophysical insights into the mitochondrial Rieske iron-sulfur protein from genetic suppressor analysis in C. elegans.

    PubMed

    Jafari, Gholamali; Wasko, Brian M; Kaeberlein, Matt; Crofts, Antony R

    2016-01-01

    Several intragenic mutations suppress the C. elegans isp-1(qm150) allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the "tether" span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with the qm150 allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a "spring-loaded" mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical "spring") favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the "spring." In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen in isp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity in C. elegans from the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a "canary" function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells. PMID:27383074

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

    PubMed

    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. PMID:26628000

  15. New functional and biophysical insights into the mitochondrial Rieske iron-sulfur protein from genetic suppressor analysis in C. elegans.

    PubMed

    Jafari, Gholamali; Wasko, Brian M; Kaeberlein, Matt; Crofts, Antony R

    2016-01-01

    Several intragenic mutations suppress the C. elegans isp-1(qm150) allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the "tether" span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with the qm150 allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a "spring-loaded" mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical "spring") favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the "spring." In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen in isp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity in C. elegans from the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a "canary" function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells.

  16. Internalization of coxsackievirus A9 is mediated by {beta}2-microglobulin, dynamin, and Arf6 but not by caveolin-1 or clathrin.

    PubMed

    Heikkilä, Outi; Susi, Petri; Tevaluoto, Tuire; Härmä, Heidi; Marjomäki, Varpu; Hyypiä, Timo; Kiljunen, Saija

    2010-04-01

    Coxsackievirus A9 (CAV9) is a member of the human enterovirus B species within the Enterovirus genus of the family Picornaviridae. It has been shown to utilize alphaV integrins, particularly alphaVbeta6, as its receptors. The endocytic pathway by which CAV9 enters human cells after the initial attachment to the cell surface has so far been unknown. Here, we present a systematic study concerning the internalization mechanism of CAV9 to A549 human lung carcinoma cells. The small interfering RNA (siRNA) silencing of integrin beta6 subunit inhibited virus proliferation, confirming that alphaVbeta6 mediates the CAV9 infection. However, siRNAs against integrin-linked signaling molecules, such as Src, Fyn, RhoA, phosphatidylinositol 3-kinase, and Akt1, did not reduce CAV9 proliferation, suggesting that the internalization of the virus does not involve integrin-linked signaling events. CAV9 endocytosis was independent of clathrin or caveolin-1 but was restrained by dynasore, an inhibitor of dynamin. The RNA interference silencing of beta2-microglobulin efficiently inhibited virus infection and caused CAV9 to accumulate on the cell surface. Furthermore, CAV9 infection was found to depend on Arf6 as both silencing of this molecule by siRNA and the expression of a dominant negative construct resulted in decreased virus infection. In conclusion, the internalization of CAV9 to A549 cells follows an endocytic pathway that is dependent on integrin alphaVbeta6, beta2-microglobulin, dynamin, and Arf6 but independent of clathrin and caveolin-1.

  17. Introduction of Caveolae Structural Proteins into the Protozoan Toxoplasma Results in the Formation of Heterologous Caveolae but Not Caveolar Endocytosis

    PubMed Central

    Lige, Bao; Sonda, Sabrina; Joiner, Keith A.; Coppens, Isabelle

    2012-01-01

    Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae ‘precursors’ in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma. PMID:23272165

  18. Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3.

    PubMed

    Zhang, Chao; Liu, Xiao-juan; Wu, Kang-cheng; Zheng, Lu-Ping; Ding, Zuo-mei; Li, Fei; Zou, Peng; Yang, Liang; Wu, Jian-guo; Wu, Zu-jian

    2015-11-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana plants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells from Nicotiana benthamiana. PMID:26296721

  19. Stress-induced phosphorylation of caveolin-1 and p38, and down-regulation of EGFr and ERK by the dietary lectin jacalin in two human carcinoma cell lines.

    PubMed

    Sahasrabuddhe, Anagh A; Ahmed, Neesar; Krishnasastry, M V

    2006-01-01

    We have examined the A431 (human epidermoid carcinoma) and HT29 (human colorectal carcinoma) cellular responses evoked by lectins of dietary origin, Jacalin of Artocarpus integrifolia (native jacalin; nJacalin), peanut agglutinin (PNA) of Arachis hypogea, and recombinant single-chain jacalin (rJacalin), which has the same protein backbone but approximately 100-fold less affinity for carbohydrates than nJacalin. All three lectins (nJacalin, rJacalin, and PNA) are cycotoxic inhibitors of proliferation of A431 cells. However, cells recover once jacalin but not PNA have been removed from the growth medium. Treatment of nJacalin results in morphologically visible cell rounding while retaining the membrane integrity when treated at 40 microg ml(-1), but treatment with PNA did not induce such changes. The observed cell rounding was found to be due to stress as the phosphorylation of caveolin-1 (at tyr14), p38 but not c-Jun N-terminal kinase were up-regulated, while PNA did not up-regulate the phosphorylation of the same. Jacalin also down-regulated the phosphorylation of the epidermal growth factor receptor and extracellular signal regulated kinase in contrast to PNA, which failed to down-regulate the same. Confocal microscopic studies reveal that jacalin is not internalized, unlike the lectin of Agaricus bisporous. Analysis of the proteins that bind to an nJacalin-sepharose column revealed the binding of six to eight proteins, and significant among them is a protein at approximately 110 kDa, which appears to be oxygen-regulated protein 150 (ORP150) (endoplasmic reticulum chaperone) as identified by its isoelectric point, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis. This 110-kDa band is detectable with anti-Hsp70 antibody because ORP150 has homology with Hsp70. Confocal microscopic studies reveal the presence of Hsp70-like proteins on the surface of A431 cells as revealed by immunostaining with anti-Hsp70

  20. In vitro incorporation of nonnatural amino acids into protein using tRNACys-derived opal, ochre, and amber suppressor tRNAs

    PubMed Central

    Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E.; Skach, William R.

    2010-01-01

    Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNACys that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

  1. In vitro incorporation of nonnatural amino acids into protein using tRNA(Cys)-derived opal, ochre, and amber suppressor tRNAs.

    PubMed

    Gubbens, Jacob; Kim, Soo Jung; Yang, Zhongying; Johnson, Arthur E; Skach, William R

    2010-08-01

    Amber suppressor tRNAs are widely used to incorporate nonnatural amino acids into proteins to serve as probes of structure, environment, and function. The utility of this approach would be greatly enhanced if multiple probes could be simultaneously incorporated at different locations in the same protein without other modifications. Toward this end, we have developed amber, opal, and ochre suppressor tRNAs derived from Escherichia coli, and yeast tRNA(Cys) that incorporate a chemically modified cysteine residue with high selectivity at the cognate UAG, UGA, and UAA stop codons in an in vitro translation system. These synthetic tRNAs were aminoacylated in vitro, and the labile aminoacyl bond was stabilized by covalently attaching a fluorescent dye to the cysteine sulfhydryl group. Readthrough efficiency (amber > opal > ochre) was substantially improved by eRF1/eRF3 inhibition with an RNA aptamer, thus overcoming an intrinsic hierarchy in stop codon selection that limits UGA and UAA termination suppression in higher eukaryotic translation systems. This approach now allows concurrent incorporation of two different modified amino acids at amber and opal codons with a combined apparent readthrough efficiency of up to 25% when compared with the parent protein lacking a stop codon. As such, it significantly expands the possibilities for incorporating nonnative amino acids for protein structure/function studies. PMID:20581130

  2. The CUG-initiated larger form coat protein of Chinese wheat mosaic virus binds to the cysteine-rich RNA silencing suppressor.

    PubMed

    Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Ratti, Claudio; Chen, Jianping

    2013-10-01

    Some viruses use alternative translation initiation at non-AUG codons as a strategy to produce multiple proteins during gene expression. Here we show that, using this strategy, Chinese wheat mosaic virus (CWMV; Furovirus) expresses a larger form of coat protein (N-ext/CP) in infected plants. Site-directed mutagenesis and transient expression analysis confirmed that CWMV N-ext/CP is initiated at an upstream in-frame CUG codon at nucleotide position 207-209 of RNA 2, which adds a 39 amino acid (aa) N-terminal extension to the major CP. Interestingly, in planta and in vitro analyses indicated that CWMV N-ext/CP but not CP interacts with the CWMV cysteine-rich protein (CRP), an RNA silencing suppressor. We further determined that the N-terminal 39 aa extension, particularly the 10 aa region immediately upstream of the major CP coding region is responsible for the interaction of N-ext/CP with CRP. In an Agrobacterium co-infiltration assay, co-expression with N-ext/CP did not affect CRP silencing suppression activity. Thus the alternative translation initiation at a CUG codon provides the CWMV N-ext/CP with the ability to bind to the viral silencing suppressor.

  3. A nucleolar protein, H19 opposite tumor suppressor (HOTS), is a tumor growth inhibitor encoded by a human imprinted H19 antisense transcript

    PubMed Central

    Onyango, Patrick; Feinberg, Andrew P.

    2011-01-01

    The H19 gene, which localizes within a chromosomal region on human chromosome 11p15 that is commonly lost in Wilms tumor (WT), encodes an imprinted untranslated RNA. However, the biological significance of the H19 noncoding transcript remains unresolved because replacement of the RNA transcript with a neocassette has no obvious phenotypic effect. Here we show that the human H19 locus also encodes a maternally expressed, translated gene, antisense to the known H19 transcript, which is conserved in primates. This gene, termed HOTS for H19 opposite tumor suppressor, encodes a protein that localizes to the nucleus and nucleolus and that interacts with the human enhancer of rudimentary homolog (ERH) protein. WTs that show loss of heterozygosity of 11p15 or loss of imprinting of IGF2 also silence HOTS (7/7 and 10/10, respectively). Overexpression of HOTS inhibits Wilms, rhabdoid, rhabdomyosarcoma, and choriocarcinoma tumor cell growth, and silencing HOTS by RNAi increases in vitro colony formation and in vivo tumor growth. These results demonstrate that the human H19 locus harbors an imprinted gene encoding a tumor suppressor protein within the long-sought WT2 locus. PMID:21940503

  4. The 1.35 A resolution structure of the phosphatase domain of the suppressor of T cell receptor signaling protein in complex with sulfate

    SciTech Connect

    Jakoncic, J.; Sondgeroth, B.; Carpino, N.; Nassar, N.

    2010-04-19

    The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis-Menten constants at acidic pH.

  5. Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection

    PubMed Central

    Zhou, Tao; Murphy, Alex M.; Lewsey, Mathew G.; Westwood, Jack H.; Zhang, Heng-Mu; González, Inmaculada; Canto, Tomás

    2014-01-01

    The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1. PMID:24633701

  6. The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses.

    PubMed

    Shimura, Hanako; Masuta, Chikara; Yoshida, Naoto; Sueda, Kae; Suzuki, Masahiko

    2013-08-01

    Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

  7. Exercise-Induced Changes in Caveolin-1, Depletion of Mitochondrial Cholesterol, and the Inhibition of Mitochondrial Swelling in Rat Skeletal Muscle but Not in the Liver.

    PubMed

    Flis, Damian Jozef; Olek, Robert Antoni; Kaczor, Jan Jacek; Rodziewicz, Ewa; Halon, Malgorzata; Antosiewicz, Jedrzej; Wozniak, Michal; Gabbianelli, Rosita; Ziolkowski, Wieslaw

    2016-01-01

    The reduction in cholesterol in mitochondria, observed after exercise, is related to the inhibition of mitochondrial swelling. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and is required by various signalling pathways. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the mitochondrial Cav-1 concentration; additionally, we identified the results of these changes as they relate to the induction of changes in the mitochondrial swelling and cholesterol in rat skeletal muscle and liver. Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their quadriceps femoris muscles and livers were immediately removed for experimentation. The exercise protocol caused an increase in the Cav-1 concentration in crude muscle mitochondria; this was related to a reduction in the cholesterol level and an inhibition of mitochondrial swelling. There were no changes in rat livers, with the exception of increased markers of oxidative stress in mitochondria. These data indicate the possible role of Cav-1 in the adaptive change in the rat muscle mitochondria following exercise. PMID:26839631

  8. Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

    PubMed Central

    Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

    2016-01-01

    Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

  9. Evidence that noncoding RNA dutA is a multicopy suppressor of Dictyostelium discoideum STAT protein Dd-STATa.

    PubMed

    Shimada, Nao; Kawata, Takefumi

    2007-06-01

    Dd-STATa, a Dictyostelium discoideum homologue of metazoan STAT transcription factors, is necessary for culmination. We created a mutant strain with partial Dd-STATa activity and used it to screen for unlinked suppressor genes. We screened approximately 450,000 clones from a slug-stage cDNA library for their ability to rescue the culmination defect when overexpressed. There were 12 multicopy suppressors of Dd-STATa, of which 4 encoded segments of a known noncoding RNA, dutA. Expression of dutA is specific to the pstA zone, the region where Dd-STATa is activated. In suppressed strains the expression patterns of several putative Dd-STATa target genes become similar to the wild-type strain. In addition, the amount of the tyrosine-phosphorylated form of Dd-STATa is significantly increased in the suppressed strain. These results indicate that partial copies of dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an unknown mechanism.

  10. ATP13A3 and caveolin-1 as potential biomarkers for difluoromethylornithine-based therapies in pancreatic cancers

    PubMed Central

    Madan, Meenu; Patel, Arjun; Skruber, Kristen; Geerts, Dirk; Altomare, Deborah A; IV, Otto Phanstiel

    2016-01-01

    The purpose of this paper was to better understand the role of polyamine transport in pancreatic cancers.This paper identifies potential biomarkers for assessing the relative tumor commitment to polyamine biosynthesis or transport. Cell lines with low polyamine import activity and low ATP13A3 protein levels appear committed to polyamine biosynthesis and required high concentrations of the polyamine biosynthesis inhibitor, difluoromethylornithine (DFMO) to inhibit their growth (e.g., AsPC-1 and Capan 1). In contrast, cell lines with high polyamine import activity and high ATP13A3 protein expression (e.g., L3.6pl) demonstrated a commitment to polyamine transport and required lower DFMO concentrations to inhibit their growth. Pancreatic cancer cell lines which were most sensitive to DFMO also gave the highest EC50 values for the polyamine transport inhibitors (PTIs) tested indicating that more PTI was needed to inhibit the active polyamine transport systems of these cell lines. Most significant is that the combination therapy of DFMO+PTI was efficacious against both cell types with the PTI showing low efficacy in cell lines with low polyamine transport activity and high efficacy in cell lines with high polyamine transport activity. High ATP13A3 protein expression and moderate to low Cav-1 protein expression was shown to be predictive of tumors which effectively escape DFMO via polyamine import. In summary, this report demonstrates for the first time the role of ATP13A3 in polyamine transport and its use as a potential biomarker along with Cav-1 to select tumors most susceptible to DFMO. These findings may help stratify patients in the ongoing clinical trials with DFMO-based therapies and help predict tumor response. PMID:27429841

  11. Gene therapy of c-myc suppressor FUSE-binding protein-interacting repressor by Sendai virus delivery prevents tracheal stenosis.

    PubMed

    Mizokami, Daisuke; Araki, Koji; Tanaka, Nobuaki; Suzuki, Hiroshi; Tomifuji, Masayuki; Yamashita, Taku; Ueda, Yasuji; Shimada, Hideaki; Matsushita, Kazuyuki; Shiotani, Akihiro

    2015-01-01

    Acquired tracheal stenosis remains a challenging problem for otolaryngologists. The objective of this study was to determine whether the Sendai virus (SeV)-mediated c-myc suppressor, a far upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), modulates wound healing of the airway mucosa, and whether it prevents tracheal stenosis in an animal model of induced mucosal injury. A fusion gene-deleted, non-transmissible SeV vector encoding FIR (FIR-SeV/ΔF) was prepared. Rats with scraped airway mucosae were administered FIR-SeV/ΔF through the tracheostoma. The pathological changes in the airway mucosa and in the tracheal lumen were assessed five days after scraping. Untreated animals showed hyperplasia of the airway epithelium and a thickened submucosal layer with extensive fibrosis, angiogenesis, and collagen deposition causing lumen stenosis. By contrast, the administration of FIR-SeV/ΔF decreased the degree of tracheal stenosis (P < 0.05) and improved the survival rate (P < 0.05). Immunohistochemical staining showed that c-Myc expression was downregulated in the tracheal basal cells of the FIR-SeV/ΔF-treated animals, suggesting that c-myc was suppressed by FIR-SeV/ΔF in the regenerating airway epithelium of the injured tracheal mucosa. The airway-targeted gene therapy of the c-myc suppressor FIR, using a recombinant SeV vector, prevented tracheal stenosis in a rat model of airway mucosal injury.

  12. The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells

    PubMed Central

    Song, Li-Bing; Li, Jun; Liao, Wen-Ting; Feng, Yan; Yu, Chun-Ping; Hu, Li-Juan; Kong, Qing-Li; Xu, Li-Hua; Zhang, Xing; Liu, Wan-Li; Li, Man-Zhi; Zhang, Ling; Kang, Tie-Bang; Fu, Li-Wu; Huang, Wen-Lin; Xia, Yun-Fei; Tsao, Sai Wah; Li, Mengfeng; Band, Vimla; Band, Hamid; Shi, Qing-Hua; Zeng, Yi-Xin; Zeng, Mu-Sheng

    2009-01-01

    The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1–mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3β signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1–silenced cells, indicating that PTEN might be a major mediator of Bmi-1–induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer. PMID:19884659

  13. Zinc-Finger Protein 545 Inhibits Cell Proliferation as a Tumor Suppressor through Inducing Apoptosis and is Disrupted by Promoter Methylation in Breast Cancer

    PubMed Central

    Luo, Xinrong; Li, Chunhong; Li, Qianqian; Peng, Weiyan; Li, Lili; Li, Shuman; Wang, Zhenyu; Tang, Liping; Ren, Guosheng; Tao, Qian

    2014-01-01

    Krüppel-associated box-containing zinc finger proteins (KRAP-ZFPs) are well recognized as key regulators of transcription, which play a crucial role in the regulation of cell proliferation, differentiation, apoptosis and tumorigenesis. We previously identified a KRAP-ZFP protein ZNF545 acting as a tumor suppressor involved in tumor pathogenesis. However, its expression and biological function in breast cancer remain elusive. In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2−) breast tumor tissues compared with paired adjacent non-tumor tissues. We further examined its expression and methylation in breast cancer cell lines by semi-quantitative RT-PCR and methylation-specific PCR. We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles. ZNF545 methylation was detected in 29% of breast tumor tissues, but not in normal breast tissues, suggesting tumor-specific methylation of ZNF545 in breast cancer. Ectopic expression of ZNF545 in MCF7 cells inhibited cell proliferation through inducing cell cycle G0/G1 arrest and apoptosis, thus as a tumor suppressor. Moreover, ZNF545 upregulated mRNA and protein levels of c-Jun/AP1, BAX, p53 and Caspase 3. Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer. PMID:25360542

  14. Suppressor Screens in Arabidopsis.

    PubMed

    Li, Xin; Zhang, Yuelin

    2016-01-01

    Genetic screens have proven to be a useful tool in the dissection of biological processes in plants. Specifically, suppressor screens have been widely used to study signal transduction pathways. Here we provide a detailed protocol for ethyl methanesulfonate (EMS) mutagenesis used in our suppressor screens in Arabidopsis and discuss the basic principles behind suppressor screen design and downstream analyses. PMID:26577776

  15. Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules.

    PubMed

    Xu, Sulei; Zhou, Xueping; Yuan, Dong; Xu, Yanchun; He, Pingnian

    2013-11-15

    Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.

  16. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

    PubMed Central

    Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

  17. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

    PubMed

    Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

  18. EphA2-Induced Angiogenesis in Ewing Sarcoma Cells Works through bFGF Production and Is Dependent on Caveolin-1

    PubMed Central

    Sáinz-Jaspeado, Miguel; Huertas-Martinez, Juan; Lagares-Tena, Laura; Martin Liberal, Juan; Mateo-Lozano, Silvia; de Alava, Enrique; de Torres, Carmen; Mora, Jaume; del Muro, Xavier Garcia; Tirado, Oscar M.

    2013-01-01

    Angiogenesis is the result of the combined activity of the tumor microenvironment and signaling molecules. The angiogenic switch is represented as an imbalance between pro- and anti-angiogenic factors and is a rate-limiting step in the development of tumors. Eph receptor tyrosine kinases and their membrane-anchored ligands, known as ephrins, constitute the largest receptor tyrosine kinase (RTK) subfamily and are considered a major family of pro-angiogenic RTKs. Ewing sarcoma (EWS) is a highly aggressive bone and soft tissue tumor affecting children and young adults. As other solid tumors, EWS are reliant on a functional vascular network for the delivery of nutrients and oxygen and for the removal of waste. Based on the biological roles of EphA2 in promoting angiogenesis, we explored the functional role of this receptor and its relationship with caveolin-1 (CAV1) in EWS angiogenesis. We demonstrated that lack of CAV1 results in a significant reduction in micro vascular density (MVD) on 3 different in vivo models. In vitro, this phenomenon correlated with inactivation of EphA2 receptor, lack of AKT response and downregulation of bFGF. We also demonstrated that secreted bFGF from EWS cells acted as chemoattractant for endothelial cells. Furthermore, interaction between EphA2 and CAV1 was necessary for the right localization and signaling of the receptor to produce bFGF through AKT and promote migration of endothelial cells. Finally, introduction of a dominant-negative form of EphA2 into EWS cells mostly reproduced the effects occurred by CAV1 silencing, strongly suggesting that the axis EphA2-CAV1 participates in the promotion of endothelial cell migration toward the tumors favoring EWS angiogenesis. PMID:23951165

  19. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

    PubMed

    Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

  20. Caveolin-1 expression is variably displayed in astroglial-derived tumors and absent in oligodendrogliomas: concrete premises for a new reliable diagnostic marker in gliomas.

    PubMed

    Cassoni, Paola; Senetta, Rebecca; Castellano, Isabella; Ortolan, Erika; Bosco, Martino; Magnani, Ivana; Ducati, Alessandro

    2007-05-01

    Caveolins are basic constituents of flask-shaped cell membrane microdomains (caveolae), which are involved in many cell functions, including signalling, trafficking, and cellular growth control. The distribution of caveolae within the normal brain and in brain tumors is controversial. In the present study, we describe the expression of caveolin-1 (cav-1) in 64 brain tumors of different grade, of either astroglial or oligodendroglial origin. All studied astrocitomas of any grade (from II to IV) were cav-1 positive, displaying staining patterns and intensity specifically associated to the different tumor grades. In all glioblastomas and gliosarcomas, cav-1 staining was extremely intense, typically localized at the cell membrane and recognized a variable percentage of cells, including the majority of spindle cells and palisade-oriented perinecrotic cells. In anaplastic astrocytomas, a less intense membrane staining or a cytoplasmic dotlike immunoreactivity were present, the latter being almost the exclusive pattern observed in diffuse astrocitomas grade II. In contrast to astroglial tumors, the striking totality of grade II oligodendrogliomas and the large majority of grade III were lacking cav-1 expression. Interestingly, a cav-1 distribution overlapping the pattern described in tissues was observed also in primary cell cultures of human glioblastomas and astrocytomas, and also in one established glioblastoma cell line (U251 MG), analyzed by means of confocal microscopy and flow cytometry. In conclusion, among astroglial tumors cav-1 expression varies in distribution, pattern, and intensity specifically according to tumor types and grades. The association between tumor progression and a more structured membranous pattern of cav-1 expression could suggest the hypothesis of a neoplastic shift towards a mesenchymal phenotype, whose behavioral and biologic significance worth further studies. Finally, the lack of cav-1 immunoreactivity in oligodendrogliomas suggests its

  1. Caveolin-1-dependent activation of the metalloprotease TACE/ADAM17 by TGF-β in hepatocytes requires activation of Src and the NADPH oxidase NOX1.

    PubMed

    Moreno-Càceres, Joaquim; Mainez, Jèssica; Mayoral, Rafael; Martín-Sanz, Paloma; Egea, Gustavo; Fabregat, Isabel

    2016-04-01

    Transforming growth factor-β (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, the balance between which decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β. We have previously shown that caveolin-1 (CAV1) is required for activation of the metalloprotease tumour necrosis factor (TNF)-α-converting enzyme/a disintegrin and metalloproteinase 17 (TACE/ADAM17), and hence transactivation of the EGFR pathway. The specific mechanism by which TACE/ADAM17 is activated has not yet been determined. Here we show that TGF-β induces phosphorylation of sarcoma kinase (Src) in hepatocytes, a process that is impaired in Cav1(-/-) hepatocytes, coincident with a decrease in phosphorylated Src in detergent-resistant membrane fractions. TGF-β-induced activation of TACE/ADAM17 and EGFR phosphorylation were blocked using the Src inhibitor PP2. Cav1(+/+) hepatocytes showed early production of reactive oxygen species (ROS) induced by TGF-β, which was not seen in Cav1(-/-) cells. Production of ROS was inhibited by both the NADPH oxidase 1 (NOX1) inhibitor STK301831 and NOX1 knock-down, which also impaired TACE/ADAM17 activation and thus EGFR phosphorylation. Finally, neither STK301831 nor NOX1 silencing impaired Src phosphorylation, but PP2 blocked early ROS production, showing that Src is involved in NOX1 activation. As expected, inhibition of Src or NOX1 increased TGF-β-induced cell death in Cav1(+/+) cells. In conclusion, CAV1 is required for TGF-β-mediated activation of TACE/ADAM17 through a mechanism that involves phosphorylation of Src and NOX1-mediated ROS production.

  2. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    PubMed

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  3. Arabidopsis CULLIN4-Damaged DNA Binding Protein 1 Interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA Complexes to Regulate Photomorphogenesis and Flowering Time[C][W

    PubMed Central

    Chen, Haodong; Huang, Xi; Gusmaroli, Giuliana; Terzaghi, William; Lau, On Sun; Yanagawa, Yuki; Zhang, Yu; Li, Jigang; Lee, Jae-Hoon; Zhu, Danmeng; Deng, Xing Wang

    2010-01-01

    CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) possesses E3 ligase activity and promotes degradation of key factors involved in the light regulation of plant development. The finding that CULLIN4 (CUL4)-Damaged DNA Binding Protein1 (DDB1) interacts with DDB1 binding WD40 (DWD) proteins to act as E3 ligases implied that CUL4-DDB1 may associate with COP1-SUPPRESSOR OF PHYA (SPA) protein complexes, since COP1 and SPAs are DWD proteins. Here, we demonstrate that CUL4-DDB1 physically associates with COP1-SPA complexes in vitro and in vivo, likely via direct interaction of DDB1 with COP1 and SPAs. The interactions between DDB1 and COP1, SPA1, and SPA3 were disrupted by mutations in the WDXR motifs of MBP-COP1, His-SPA1, and His-SPA3. CUL4 cosuppression mutants enhanced weak cop1 photomorphogenesis and flowered early under short days. Early flowering of short day–grown cul4 mutants correlated with increased FLOWERING LOCUS T transcript levels, whereas CONSTANS transcript levels were not altered. De-etiolated1 and COP1 can bind DDB1 and may work with CUL4-DDB1 in distinct complexes, but they mediate photomorphogenesis in concert. Thus, a series of CUL4-DDB1-COP1-SPA E3 ligase complexes may mediate the repression of photomorphogenesis and, possibly, of flowering time. PMID:20061554

  4. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  5. Tumor suppressor Smad4 mediates downregulation of the anti-adhesive invasion-promoting matricellular protein SPARC: Landscaping activity of Smad4 as revealed by a "secretome" analysis.

    PubMed

    Volmer, Martin W; Radacz, Yvonne; Hahn, Stephan A; Klein-Scory, Susanne; Stühler, Kai; Zapatka, Marc; Schmiegel, Wolff; Meyer, Helmut E; Schwarte-Waldhoff, Irmgard

    2004-05-01

    We have demonstrated previously that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas cell growth in vitro was not affected. Here we show that Smad4-positive and Smad4-negative SW480 cells deposit extracellular matrices in tissue culture which are functionally different with respect to their adhesiveness. We present a "differential secretomics analysis" as the most direct approach to identify the underlying alterations. The protein composition of conditioned media produced by Smad4-positive and Smad4-negative SW480 cells was compared by two-dimensional (2-D) gel electrophoresis. A major group of protein spots was detected in media derived from Smad4-negative cells, only, which were identified as "secreted protein, acidic and rich in cysteins" (SPARC) by mass spectrometry. SPARC expression in SW480 cells was suppressed by Smad4 at the level of transcription. SPARC is a glycoprotein of the extracellular matrix, characterized as an anti-adhesive and invasion-promoting protein. Differential secretomics appeared as a powerful method to identify a novel Smad4 target gene, which may be one of the players involved in reduced adhesiveness of extracellular matrices and thus consistent with Smad4's emerging role as an invasion suppressor.

  6. Analysis of promoter hypermethylation of death-associated protein kinase and p16 tumor suppressor genes in actinic keratoses and squamous cell carcinomas of the skin.

    PubMed

    Tyler, Lisa N; Ai, Lingbao; Zuo, Chunlai; Fan, Chun-Yang; Smoller, Bruce R

    2003-07-01

    Death-associated protein kinase is a serine/threonine protein kinase implicated in promoting apoptosis and tumor suppression, whereas p16 is a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and 6 activity and arrests the cell cycle in the G1 phase. Hypermethylation of death-associated protein kinase or p16 gene with resultant gene inactivation has been described in a wide variety of human cancers. Promoter methylation of the death-associated protein kinase and p16 gene has been found in about 55% and 30% cases of head and neck squamous cell carcinoma respectively but has not yet been analyzed in cutaneous premalignant and malignant lesions. A total of 33 cases were examined for evidence of death-associated protein kinase and p16 hypermethylation and these consist of 9 cases of spongiotic dermatitis as nonneoplastic skin control, 9 cases of actinic keratosis, 8 cases of squamous cell carcinoma in situ, and 7 cases of invasive squamous cell carcinoma. Death-associated protein kinase promoter methylation was detected in 1 case of squamous cell carcinoma in situ and 1 case of nonneoplastic skin control but none of the cases of invasive squamous cell carcinoma or actinic keratosis. P16 promoter methylation was detected in 1 case of invasive squamous cell carcinoma and 1 case of nonneoplastic skin control but none of the cases of squamous cell carcinoma in situ or actinic keratosis. Promoter hypermethylation of the death-associated protein kinase and p16 genes does not appear to play an important role in the development of cutaneous squamous cell carcinoma. The data thus suggest that the mechanisms of ultraviolet-induced cutaneous carcinomas differ from those involved in the development of head and neck squamous cell carcinoma, a malignant disease induced by tobacco and alcohol exposure.

  7. A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth.

    PubMed

    Fernandez-Zapico, Martin E; Lomberk, Gwen A; Tsuji, Shoichiro; DeMars, Cathrine J; Bardsley, Michael R; Lin, Yi-Hui; Almada, Luciana L; Han, Jing-Jing; Mukhopadhyay, Debabrata; Ordog, Tamas; Buttar, Navtej S; Urrutia, Raul

    2011-04-15

    SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter. PMID:21171965

  8. Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions.

    PubMed

    Schnerwitzki, Danny; Perner, Birgit; Hoppe, Beate; Pietsch, Stefan; Mehringer, Rebecca; Hänel, Frank; Englert, Christoph

    2014-09-01

    The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication. PMID:25014653

  9. The Tomato Cell Death Suppressor Adi3 Is Restricted to the Endosomal System in Response to the Pseudomonas syringae Effector Protein AvrPto

    PubMed Central

    Ek-Ramos, María J.; Avila, Julian; Nelson Dittrich, Anna C.; Su, Dongyin; Gray, Joel W.; Devarenne, Timothy P.

    2014-01-01

    The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance. PMID:25350368

  10. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein

    PubMed Central

    Cheng, Pingyan; Corzo, Cesar A.; Luetteke, Noreen; Yu, Bin; Nagaraj, Srinivas; Bui, Marylin M.; Ortiz, Myrna; Nacken, Wolfgang; Sorg, Clemens; Vogl, Thomas; Roth, Johannes; Gabrilovich, Dmitry I.

    2008-01-01

    Accumulation of myeloid-derived suppressor cells (MDSCs) associated with inhibition of dendritic cell (DC) differentiation is one of the major immunological abnormalities in cancer and leads to suppression of antitumor immune responses. The molecular mechanism of this phenomenon remains unclear. We report here that STAT3-inducible up-regulation of the myeloid-related protein S100A9 enhances MDSC production in cancer. Mice lacking this protein mounted potent antitumor immune responses and rejected implanted tumors. This effect was reversed by administration of wild-type MDSCs from tumor-bearing mice to S100A9-null mice. Overexpression of S100A9 in cultured embryonic stem cells or transgenic mice inhibited the differentiation of DCs and macrophages and induced accumulation of MDSCs. This study demonstrates that tumor-induced up-regulation of S100A9 protein is critically important for accumulation of MDSCs and reveals a novel molecular mechanism of immunological abnormalities in cancer. PMID:18809714

  11. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  12. Rational optimization of amber suppressor tRNAs toward efficient incorporation of a non-natural amino acid into protein in a eukaryotic wheat germ extract.

    PubMed

    Ogawa, Atsushi; Namba, Yuki; Gakumasawa, Mai

    2016-03-01

    Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 μg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE. PMID:26832824

  13. A Novel Method for Gene-Specific Enhancement of Protein Translation by Targeting 5’UTRs of Selected Tumor Suppressors

    PubMed Central

    Master, Adam; Wójcicka, Anna; Giżewska, Kamilla; Popławski, Piotr; Williams, Graham R.; Nauman, Alicja

    2016-01-01

    Background Translational control is a mechanism of protein synthesis regulation emerging as an important target for new therapeutics. Naturally occurring microRNAs and synthetic small inhibitory RNAs (siRNAs) are the most recognized regulatory molecules acting via RNA interference. Surprisingly, recent studies have shown that interfering RNAs may also activate gene transcription via the newly discovered phenomenon of small RNA-induced gene activation (RNAa). Thus far, the small activating RNAs (saRNAs) have only been demonstrated as promoter-specific transcriptional activators. Findings We demonstrate that oligonucleotide-based trans-acting factors can also specifically enhance gene expression at the level of protein translation by acting at sequence-specific targets within the messenger RNA 5’-untranslated region (5’UTR). We designed a set of short synthetic oligonucleotides (dGoligos), specifically targeting alternatively spliced 5’UTRs in transcripts expressed from the THRB and CDKN2A suppressor genes. The in vitro translation efficiency of reporter constructs containing alternative TRβ1 5’UTRs was increased by up to more than 55-fold following exposure to specific dGoligos. Moreover, we found that the most folded 5’UTR has higher translational regulatory potential when compared to the weakly folded TRβ1 variant. This suggests such a strategy may be especially applied to enhance translation from relatively inactive transcripts containing long 5’UTRs of complex structure. Significance This report represents the first method for gene-specific translation enhancement using selective trans-acting factors designed to target specific 5’UTR cis-acting elements. This simple strategy may be developed further to complement other available methods for gene expression regulation including gene silencing. The dGoligo-mediated translation-enhancing approach has the potential to be transferred to increase the translation efficiency of any suitable target gene

  14. Activation of tumor suppressor protein p53 is required for Theiler's murine encephalomyelitis virus-induced apoptosis in M1-D macrophages.

    PubMed

    Son, Kyung-No; Pugazhenthi, Subbiah; Lipton, Howard L

    2009-10-01

    Theiler's murine encephalomyelitis virus (TMEV) is a highly cytolytic picornavirus that persists in the mouse central nervous system (CNS) largely in macrophages with infection maintained by macrophage-to-macrophage spread. Infected macrophages in the CNS undergo apoptosis. We recently showed that M1-D macrophages infected with the low-neurovirulence TMEV BeAn virus became apoptotic through the mitochondrial pathway that is Bax mediated. Our present analyses of the molecular events and signaling pathway(s) culminating in the mitochondrial outer membrane permeabilization that initiates the caspase cascade and apoptosis of BeAn virus-infected M1-D macrophages revealed activation of p38 mitogen-activated protein kinase by 2 to 3 h postinfection (p.i.), followed by phosphorylation of tumor suppressor protein p53 Ser 15 at 3 to 6 h p.i., stabilizing p53 levels until 6 h p.i. Activated p53 upregulated the transcription of proapoptotic puma and noxa genes at 2 to 4 h p.i. and their BH3-only protein expression, followed by the loss of detectable prosurvival Mcl-1 and A1 proteins at 4 to 10 h p.i. Degradation of the prosurvival proteins is known to release Bax, which forms homo-oligomers and translocates into and permeabilizes the mitochondrial outer membrane. Inhibition of phospho-p38 by two specific inhibitors, SB203580 and BIRB796, led to a significant decrease in apoptosis at 10 h p.i., with no effect on virus titers (only SB203580 tested). Together, these data indicate that p53 activation is required for the induction of apoptosis in infected M1-D cells.

  15. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

    SciTech Connect

    Yuan Baozhu Jefferson, Amy M.; Millecchia, Lyndell; Popescu, Nicholas C.; Reynolds, Steven H.

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.

  16. Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer

    PubMed Central

    1994-01-01

    Using a monoclonal antibody, we have identified and characterized a previously unknown cell surface protein in chicken that we call neogenin and have determined its primary sequence. The deduced amino acid sequence and structure of neogenin characterize it as a member of the immunoglobulin (Ig) superfamily. Based on amino acid sequence similarities, neogenin is closely related to the human tumor suppressor molecule DCC (deleted in colorectal cancer). Neogenin and DCC define a subgroup of Ig superfamily proteins structurally distinct from other Ig molecules such as N-CAM, Ng-CAM, and Bravo/Nr-CAM. As revealed by antibody staining of tissue sections and Western blots, neogenin expression correlates with the onset of neuronal differentiation. Neogenin is also found on cells in the lower gastrointestinal tract of embryonic chickens. DCC has been observed in human neural tissues and has been shown to be essential for terminal differentiation of specific cell types in the adult human colon. These parallels suggest that neogenin, like DCC, is functionally involved in the transition from cell proliferation to terminal differentiation of specific cell types. Since neogenin is expressed on growing neurites and downregulated at termination of neurite growth, it may also play an important role in many of the complex functional aspects of neurite extension and intercellular signaling. PMID:7806578

  17. Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC

    PubMed Central

    Zhou, Weihua; Liu, Feng; Zhang, Bin; Zhu, Min; Yang, Qin; Zeng, Yayue; Sun, Yang; Sun, Shuming; Wang, Yanpeng; Zhang, Yibin; Weng, Haibo; Chen, Lixiang; Ye, Mao; An, Xiuli; Liu, Jing

    2016-01-01

    Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N. PMID:26575790

  18. RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.

    PubMed

    Poria, D K; Guha, A; Nandi, I; Ray, P S

    2016-03-31

    Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3'-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a 'miRNA sponge' to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.

  19. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells.

    PubMed

    Yuan, Bao-Zhu; Jefferson, Amy M; Millecchia, Lyndell; Popescu, Nicholas C; Reynolds, Steven H

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer. PMID:17888903

  20. Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    PubMed Central

    Liwak, Urszula; Thakor, Nehal; Jordan, Lindsay E.; Roy, Rajat; Lewis, Stephen M.; Pardo, Olivier E.; Seckl, Michael

    2012-01-01

    Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression. PMID:22431522

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

    NASA Astrophysics Data System (ADS)

    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 < 5 å) but binds the membrane tightly with its two major domains, the C2 and

  2. Suppressor of Ty homolog-5, a novel tumor-specific human telomerase reverse transcriptase promoter-binding protein and activator in colon cancer cells

    PubMed Central

    Dong, Yong; He, Chao; Hu, Xiaotong

    2015-01-01

    The human telomerase reverse transcriptase (hTERT) promoter promotes differential hTERT gene expression in tumor cells and normal cells. However, information on the mechanisms underlying the differential hTERT transcription and induction of telomerase activity in tumor cells is limited. In the present study, suppressor of Ty homolog-5 (SPT5), a protein encoded by the SUPT5H gene, was identified as a novel tumor-specific hTERT promoter-binding protein and activator in colon cancer cells. We verified the tumor-specific binding activity of SPT5 to the hTERT promoter in vitro and in vivo and detected high expression levels of SUPT5H in colorectal cancer cell lines and primary human colorectal cancer tissues. SUPT5H was more highly expressed in colorectal cancer cases with distant metastasis than in cases without distant metastasis. Inhibition of endogenous SUPT5H expression by SUPT5H gene-specific short hairpin RNAs effectively attenuated hTERT promoter-driven green fluorescent protein (GFP) expression, whereas no detectable effects on CMV promoter-driven GFP expression in the same cells were observed. In addition, inhibition of SUPT5H expression not only effectively repressed telomerase activity, accelerated telomere shortening, and promoted cell senescence in colon cancer cells, but also suppressed cancer cell growth and migration. Our results demonstrated that SPT5 contributes to the up-regulation of hTERT expression and tumor development, and SUPT5H may potentially be used as a novel tumor biomarker and/or cancer therapeutic target. PMID:26418880

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

    PubMed

    Medvetz, Doug A; Khabibullin, Damir; Hariharan, Venkatesh; Ongusaha, Pat P; Goncharova, Elena A; Schlechter, Tanja; Darling, Thomas N; Hofmann, Ilse; Krymskaya, Vera P; Liao, James K; Huang, Hayden; Henske, Elizabeth P

    2012-01-01

    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. PMID:23139756

  4. The Arabidopsis Transcription Factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 Is a Direct Substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and Regulates Immunity1

    PubMed Central

    Kang, Sining; Yang, Fan; Li, Lin; Chen, Huamin; Chen, She; Zhang, Jie

    2015-01-01

    Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1SSAA) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1SSAA does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation. PMID:25609555

  5. The ABA-deficiency suppressor locus HAS2 encodes the PPR protein LOI1/MEF11 involved in mitochondrial RNA editing.

    PubMed

    Sechet, Julien; Roux, Camille; Plessis, Anne; Effroy, Delphine; Frey, Anne; Perreau, François; Biniek, Catherine; Krieger-Liszkay, Anja; Macherel, David; North, Helen M; Mireau, Hakim; Marion-Poll, Annie

    2015-04-01

    The hot ABA-deficiency suppressor2 (has2) mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the mitochondrial editing factor11 (MEF11), also known as lovastatin insensitive1. has2/mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hai1-1 pp2ca-1 hab1-1 abi1-2, which is impaired in four genes encoding type 2C protein phosphatases (PP2C) that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol (a gibberellin biosynthesis inhibitor) as well as mannitol and NaCl, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in mef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.

  6. Interaction of the Ras-related protein associated with diabetes Rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation

    PubMed Central

    Zhu, Jianhua; Tseng, Yu-Hua; Kantor, Jason D.; Rhodes, Christopher J.; Zetter, Bruce R.; Moyers, Julie S.; Kahn, C. Ronald

    1999-01-01

    Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP⋅Rad was also reconverted to GTP⋅Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation. PMID:10611312

  7. Heat shock protein 60 activates cytokine-associated negative regulator suppressor of cytokine signaling 3 in T cells: effects on signaling, chemotaxis, and inflammation.

    PubMed

    Zanin-Zhorov, Alexandra; Tal, Guy; Shivtiel, Shoham; Cohen, Michal; Lapidot, Tsvee; Nussbaum, Gabriel; Margalit, Raanan; Cohen, Irun R; Lider, Ofer

    2005-07-01

    Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1alpha (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1alpha and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation. PMID:15972659

  8. The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency*

    PubMed Central

    Sengupta, Satyaki; Lingnurkar, Raj; Carey, Timothy S.; Pomaville, Monica; Kar, Parimal; Feig, Michael; Wilson, Catherine A.; Knott, Jason G.; Arnosti, David N.; Henry, R. William

    2015-01-01

    The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system. PMID:25903125

  9. Kinetics of purified protein derivative (PPD) proliferation reflects underlying suppressor mechanisms revealed by limiting dilution analysis (LDA) in patients with extrapulmonary tuberculosis (TB)

    PubMed Central

    Lukey, P T; Latouf, S E; Ress, S R

    1998-01-01

    Mononuclear leucocytes from the blood (PBML) and effusion (EML) of patients undergoing pericardiocentesis were assayed for proliferative response to purified protein derivative of Mycobacterium tuberculosis (PPD). Of the 23 patients tested, 10 had culture-positive tuberculous effusions, while 13 had non-tuberculous aetiologies. Three different kinetic responses were identified: (i) accelerated responses (found in 70% of EML from patients with culture-positive tuberculous effusions); (ii) ‘flat’ responses (found in 10% of EML from patients with culture-positive tuberculous effusions); and (iii) normal kinetic responses. These differences in kinetic response may reflect underlying immune mechanisms important in the immunopathogenesis of TB. In order to address this possibility we performed LDA on a selection of patients with culture-positive extrapulmonary TB: three patients with accelerated responses, two with normal responses, and one with a ‘flat’ response. The results confirm the previously reported accumulation of PPD-specific responder cells in the effusion of patients with TB. Cell-mediated suppressor mechanisms (as shown by ‘V’-shaped LDA curves) were found in the blood of one patient and the effusion of another. In both cases ‘flat’ PPD-proliferative responses were observed. However, the LDA data also suggested the presence of in vivo mechanisms limiting the clonal burst size. Thus it appears that immune responses in extrapulmonary TB are influenced by an array of inhibitory mechanisms, modulation of which may influence the outcome of infection. PMID:9486395

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

    PubMed Central

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

    2012-01-01

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

  11. Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

    PubMed

    Chan, Ka Lam; Bakman, Inna; Marts, Amy R; Batir, Yuksel; Dowd, Terry L; Tierney, David L; Gibney, Brian R

    2014-06-16

    Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc

  12. Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

    PubMed

    Chan, Ka Lam; Bakman, Inna; Marts, Amy R; Batir, Yuksel; Dowd, Terry L; Tierney, David L; Gibney, Brian R

    2014-06-16

    Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc

  13. Placental expression of eNOS, iNOS and the major protein components of caveolae in women with pre-eclampsia.

    PubMed

    Smith-Jackson, K; Hentschke, M R; Poli-de-Figueiredo, C E; Pinheiro da Costa, B E; Kurlak, L O; Broughton Pipkin, F; Czajka, A; Mistry, H D

    2015-05-01

    Caveolae regulate many cardiovascular functions and thus could be of interest in relation to pre-eclampsia, a pregnancy specific disorder characterised by hypertension and proteinuria. We examined placental mRNA and protein expression/localisation of the caveolae components Caveolin 1-3, Cavin 1-4 as well as eNOS/iNOS in normotensive control (n = 24) and pre-eclamptic pregnancies (n = 19). Placental mRNA expression of caveolin-1, cavin 1-3, was lower and eNOS expression was increased in pre-eclampsia (P < 0.05 for all). Additionally Caveolin-1 protein expression was also reduced in pre-eclampsia (P = 0.007); this could be an adaptive response in pre-eclampsia, possibly to attenuate the oxidative stress/inflammation. PMID:25707739

  14. Design, Synthesis, and Characterization of Cyclic Peptidomimetics of the Inducible Nitric Oxide Synthase Binding Epitope That Disrupt the Protein-Protein Interaction Involving SPRY Domain-Containing Suppressor of Cytokine Signaling Box Protein (SPSB) 2 and Inducible Nitric Oxide Synthase.

    PubMed

    Harjani, Jitendra R; Yap, Beow Keat; Leung, Eleanor W W; Lucke, Andrew; Nicholson, Sandra E; Scanlon, Martin J; Chalmers, David K; Thompson, Philip E; Norton, Raymond S; Baell, Jonathan B

    2016-06-23

    SPRY domain-containing suppressor of cytokine signaling box protein (SPSB) 2-deficient macrophages have been found to exhibit prolonged expression of inducible nitric oxide synthase (iNOS) and enhanced killing of persistent pathogens, suggesting that inhibitors of the SPSB2-iNOS interaction have potential as novel anti-infectives. In this study, we describe the design, synthesis, and characterization of cyclic peptidomimetic inhibitors of the SPSB2-iNOS interaction constrained by organic linkers to improve stability and druggability. SPR, ITC, and (19)F NMR analyses revealed that the most potent cyclic peptidomimetic bound to the iNOS binding site of SPSB2 with low nanomolar affinity (KD 29 nM), a 10-fold improvement over that of the linear peptide DINNN (KD 318 nM), and showed strong inhibition of SPSB2-iNOS interaction in macrophage cell lysates. This study exemplifies a novel approach to cyclize a Type II β-turn linear peptide and provides a foundation for future development of this group of inhibitors as new anti-infectives.

  15. Inhalational anesthetics disrupt postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 domain protein interactions critical to action of several excitatory receptor channels related to anesthesia

    PubMed Central

    Tao, Feng; Chen, Qiang; Sato, Yuko; Skinner, John; Tang, Pei; Johns, Roger A.

    2015-01-01

    Background We have shown previously that inhaled anesthetics disrupt the interaction between the second postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain of postsynaptic density protein-95 (PSD-95) and the C-terminus of N-methyl-D-aspartate receptor subunits NR2A and NR2B. Our data indicate that PDZ domains may serve as a molecular target for inhaled anesthetics. However, the underlying molecular mechanisms remain to be illustrated. Methods Glutathione S-transferase pull-down assay, co-immunoprecipitation and yeast two-hybrid analysis were used to assess PDZ domain-mediated protein-protein interactions in different conditions. Nuclear magnetic resonance spectroscopy was used to investigate isoflurane-induced chemical shift changes in the PDZ1–3 domains of PSD-95. A surface plasmon resonance-based BIAcore assay was used to examine the ability of isoflurane to inhibit the PDZ domain-mediated protein-protein interactions in real time. Results Halothane and isoflurane dose dependently inhibited PDZ domain-mediated interactions between PSD-95 and Shaker-type potassium channel Kv1.4 and between α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluA2 and its interacting proteins— glutamate receptor interacting protein or protein interacting with c kinase 1. However, halothane and isoflurane had no effect on PDZ domain-mediated interactions between γ-aminobutyric acid, type B receptor and its interacting proteins. The inhaled anesthetic isoflurane mostly affected the residues close to or in the peptide binding groove of PSD-95 PDZ1 and PDZ2 (especially PDZ2), while barely affecting the peptide binding groove of PSD-95 PDZ3. Conclusion These results suggest that inhaled anesthetics interfere with PDZ domain-mediated protein-protein interactions at several receptors important to neuronal excitation, anesthesia and pain processing. PMID:25654436

  16. Genetic Modeling of PIM Proteins in Cancer: Proviral Tagging and Cooperation with Oncogenes, Tumor Suppressor Genes, and Carcinogens

    PubMed Central

    Aguirre, Enara; Renner, Oliver; Narlik-Grassow, Maja; Blanco-Aparicio, Carmen

    2014-01-01

    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 anti-cancer 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 three 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. PMID:24860787

  17. Inhibition of growth by p205: a nuclear protein and putative tumor suppressor expressed during myeloid cell differentiation.

    PubMed

    Dermott, Jonathan M; Gooya, John M; Asefa, Benyam; Weiler, Sarah R; Smith, Mark; Keller, Jonathan R

    2004-01-01

    p205 belongs to a family of interferon-inducible proteins called the IFI-200 family, which have been implicated in the regulation of cell growth and differentiation. While p205 is induced in hematopoietic stem cells during myeloid cell differentiation, its function is not known. Therefore, the aim of this study was to determine the role of p205 in regulating proliferation in hematopoietic progenitor cells and in nonhematopoietic cell lines. We found that p205 localizes to the nucleus in hematopoietic and nonhematopoietic cell lines. Transient expression of p205 in murine IL-3-dependent BaF3 and 32D-C123 progenitor cell lines inhibited IL-3-induced growth and proliferation. The closely related IFI-200 family members, p204 and p202, similarly inhibited IL-3-dependent progenitor cell proliferation. p205 also inhibited the proliferation and growth of normal hematopoietic progenitor cells. In nonhematopoietic cell lines, p205 and p204 expression inhibited NIH3T3 cell colony formation in vitro, and microinjection of p205 expression vectors into NIH3T3 fibroblasts inhibited serum-induced proliferation. We have determined the functional domains of p205 necessary for activity, which were identified as the N-terminal domain in apoptosis and interferon response (DAPIN)/PYRIN domain, and the C-terminal retinoblastoma protein (Rb)-binding motif. In addition, we have demonstrated that a putative ataxia telangiectasia, mutated (ATM) kinase phosphorylation site specifically regulates the activity of p205. Taken together, these data suggest that p205 is a potent cell growth regulator whose activity is mediated by its protein-binding domains. We propose that during myelomonocytic cell differentiation, induction of p205 expression contributes to cell growth arrest, thus allowing progenitor cells to differentiate. PMID:15342947

  18. Genetic Modeling of PIM Proteins in Cancer: Proviral Tagging and Cooperation with Oncogenes, Tumor Suppressor Genes, and Carcinogens.

    PubMed

    Aguirre, Enara; Renner, Oliver; Narlik-Grassow, Maja; Blanco-Aparicio, Carmen

    2014-01-01

    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 anti-cancer 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 three 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. PMID:24860787

  19. Molecular cloning, characterization and expression analysis of tumor suppressor protein p53 from orange-spotted grouper, Epinephelus coioides in response to temperature stress.

    PubMed

    Qi, Zeng-Hua; Liu, Yu-Feng; Luo, Sheng-Wei; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

    2013-11-01

    The tumor suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. In the present study, the cDNA of p53 from the orange-spotted grouper (Epinephelus coioides) (Ec-p53) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Ec-p53 was of 1921 bp, including an open reading frame (ORF) of 1143 bp encoding a polypeptide of 380 amino acids with predicted molecular weight of 42.3 kDa and theoretical isoelectric point of 7.0. Quantitative real-time PCR (qRT-PCR) assays revealed that Ec-p53 was ubiquitously expressed in all the examined tissues but with high levels in intestine and liver of the orange-spotted grouper. In addition, we measured the DNA damage and apoptosis in the blood cells and the percentage of dead and damaged blood cells. Our results suggest that oxidative stress and DNA damage occurred in grouper in conditions where the temperature was 15 ± 0.5 °C. Furthermore, qRT-PCR and western blot confirmed that low temperature stress induced upregulation of Ec-p53 in the mRNA and protein levels. These results suggest that low temperature-induced oxidative stress may cause DNA damage or apoptosis, and cooperatively stimulate the expression of Ec-p53, which plays a critical role in immune defense and antioxidant responses.

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

    SciTech Connect

    Cai, Liquan; Wang, Dan; Fisher, Alfred L.; Wang, Zhou

    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

  1. The tumor suppressor proteins ASPP1 and ASPP2 interact with C-Nap1 and regulate centrosome linker reassembly.

    PubMed

    Zhang, Yuanyuan; Wang, Yuqi; Wei, Youheng; Ma, Jian; Peng, Jingtao; Wumaier, Reziya; Shen, Suqin; Zhang, Pingzhao; Yu, Long

    2015-03-13

    Centrosome linker tethers interphase centrosomes together allowing them to function as a single microtubule organization center. The centrosome linker is disrupted at the onset of mitosis to ensure timely centrosome disjunction and bipolar spindle formation and is reassembled at the end of mitosis. While the mechanism controlling centrosome linker disassembly at early mitosis has been well explored, little is known about how the linker is subsequently reassembled before mitotic exit. Here we report that ASPP1 and ASPP2, two members of the apoptosis stimulating proteins of p53 (ASPP) family, are involved in centrosome linker reassembly. We showed that ASPP1/2 interacted with centrosome linker protein C-Nap1. Co-depletion of ASPP1 and ASPP2 inhibited re-association of C-Nap1 with centrosome at the end of mitosis. Moreover, ASPP1/2 facilitated the interaction between C-Nap1 and PP1α, and this interaction was significantly reduced by co-depletion of ASPP1/2. ASPP1/2 antagonized the NEK2A-mediated C-Nap1 Ser2417/2421 phosphorylation in a PP1-dependent manner. Co-depletion of ASPP1 and ASPP2 inhibited dephosphorylation of C-Nap1 (Ser2417/2421) at the end of mitosis. Based on these findings, we propose that ASPP1/2 act as PP1-targeting subunits to facilitate C-Nap1 dephosphorylation and centrosome linker reassembly at the end of mitosis. PMID:25660448

  2. The Suppressor of Hairy-Wing Protein Regulates the Tissue-Specific Expression of the Drosophila Gypsy Retrotransposon

    PubMed Central

    Smith, P. A.; Corces, V. G.

    1995-01-01

    The gypsy retrotransposon of Drosophila melanogaster causes mutations that show temporal and tissue-specific phenotypes. These mutant phenotypes can be reversed by mutations in su(Hw), a gene that also regulates the transcription of the gypsy element. Gypsy encodes a full-length 7.0-kb RNA that is expressed in the salivary gland precursors and fat body of the embryo, imaginal discs and fat body of larvae, and fat body and ovaries of adult females. The su(Hw)-binding region inserted upstream of the promoter of a lacZ reporter gene can induce β-galactosidase expression in a subset of the embryonic and larval tissues where gypsy is normally transcribed. This expression is dependent on the presence of a functional su(Hw) product, suggesting that this protein is a positive activator of gypsy transcription. Flies transformed with a construct in which the 5' LTR and leader sequences of gypsy are fused to lacZ show β-galactosidase expression in all tissues where gypsy is normally expressed, indicating that sequences other than the su(Hw)-binding site are required for proper spatial and temporal expression of gypsy. Mutations in the zinc fingers of su(Hw) affect its ability to bind DNA and to induce transcription of the lacZ reporter gene. Two other structural domains of su(Hw) also play an important role in transcriptional regulation of gypsy. Deletion of the amino-terminal acidic domain results in the loss of lacZ expression in larval fat body and adult ovaries, whereas mutations in the leucine zipper region result in an increase of lacZ expression in larval fat body and a decrease in adult ovaries. These effects might be the result of interactions of su(Hw) with activator and repressor proteins through the acidic and leucine zipper domains to produce the final pattern of tissue-specific expression of gypsy. PMID:7705625

  3. MicroRNA-326 functions as a tumor suppressor in glioma by targeting the Nin one binding protein (NOB1).

    PubMed

    Zhou, Jingxu; Xu, Tao; Yan, Yong; Qin, Rong; Wang, Hongxiang; Zhang, Xiaoping; Huang, Yan; Wang, Yuhai; Lu, Yicheng; Fu, Da; Chen, Juxiang

    2013-01-01

    Malignant glioma is the most common type of primary brain tumor in adults, characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. Alterations in the activity of the 26S proteasome have been associated with malignant glioma cells, although the specific defects have not been identified. Recently, microRNA-326 (miR-326) was shown to play an important role in glioblastoma and breast cancer, but the underlying molecular mechanisms remain unclear. In the present study, the human Nin one binding protein (NOB1) was identified as a direct target of miR-326 and a potential oncogene in human glioma. Similar to NOB1 silencing by shRNA, overexpression of miR-326 in human glioma cell lines (A172 and U373) caused cell cycle arrest at the G1 phase, delayed cell proliferation and enhanced apoptosis. MiR-326 inhibited colony formation in soft agar and decreased growth of a xenograft tumor model, suggesting that miR-326 and NOB1 are required for tumorigenesis in vitro and in vivo. Furthermore, these processes were shown to involve the MAPK pathway. NOB1 overexpression in human glioma samples was detected by Affymetrix array analysis, and NOB1 mRNA and protein levels were shown to be increased in high-grade glioma compared to low-grade glioma and normal brain tissue. Furthermore, high levels of NOB1 were associated with unfavorable prognosis of glioma patients. Taken together, these results indicate that miR-326 and NOB1 may play an important role in the development of glioma.

  4. Methylation and mRNA expression levels of P15, death-associated protein kinase, and suppressor of cytokine signaling-1 genes in multiple myeloma

    PubMed Central

    Liu, Lin; Tan, Lin; He, Zhenxin

    2016-01-01

    Objective(s): The aim of this study was to investigate the methylation status and mRNA expression levels of P15, death-associated protein kinase (DAPK), and suppressor of cytokine signaling-1 (SOCS1) genes in multiple myeloma (MM). Materials and Methods: The bone marrow samples of 54 MM patients were collected and the methylation status of the P15, DAPK, and SOCS1 gene promoter regions was determined by methylation-specific polymerase chain reaction. Automated sequencing technology was used to sequence the amplified products in order to analyze the base methylation sites. mRNA expression levels were determined using real-time fluorescent quantitative polymerase chain reaction. Results: Among the 54 MM patients, the positive methylation rates of the P15, DAPK, and SOCS1 genes were 27.78%, 18.52%, and 16.67%, respectively. The methylation results were confirmed by sequencing. The positive methylation rates of the P15, DAPK, and SOCS1 genes showed no correlation with patient gender, age, typing, staging, and grouping (P>0.05). There was no significant difference in the mRNA expression levels of the P15, DAPK, and SOCS1 genes between the MM patient group and the control group (P>0.05). Conclusions: Aberrant methylation of the P15, DAPK, and SOCS1 genes exists in MM, and these genes may play certain roles in pathogenesis of MM. There was no significant difference in mRNA expression levels between the methylated group and the non-methylated group, suggesting that these genes are regulated by other mechanisms during their transcription. PMID:27635200

  5. A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor.

    PubMed

    Lee, M; Daniels, M J; Garnett, M J; Venkitaraman, A R

    2011-07-28

    The inactivation of BRCA2, a suppressor of breast, ovarian and other epithelial cancers, triggers instability in chromosome structure and number, which are thought to arise from defects in DNA recombination and mitotic cell division, respectively. Human BRCA2 controls DNA recombination via eight BRC repeats, evolutionarily conserved motifs of ∼35 residues, that interact directly with the recombinase RAD51. How BRCA2 controls mitotic cell division is debated. Several studies by different groups report that BRCA2 deficiency affects cytokinesis. Moreover, its interaction with HMG20b, a protein of uncertain function containing a promiscuous DNA-binding domain and kinesin-like coiled coils, has been implicated in the G2-M transition. We show here that HMG20b depletion by RNA interference disturbs the completion of cell division, suggesting a novel function for HMG20b. In vitro, HMG20b binds directly to the BRC repeats of BRCA2, and exhibits the highest affinity for BRC5, a motif that binds poorly to RAD51. Conversely, the BRC4 repeat binds strongly to RAD51, but not to HMG20b. In vivo, BRC5 overexpression inhibits the BRCA2-HMG20b interaction, recapitulating defects in the completion of cell division provoked by HMG20b depletion. In contrast, BRC4 inhibits the BRCA2-RAD51 interaction and the assembly of RAD51 at sites of DNA damage, but not the completion of cell division. Our findings suggest that a novel function for HMG20b in cytokinesis is regulated by its interaction with the BRC repeats of BRCA2, and separate this unexpected function for the BRC repeats from their known activity in DNA recombination. We propose that divergent tumor-suppressive pathways regulating chromosome segregation as well as chromosome structure may be governed by the conserved BRC motifs in BRCA2.

  6. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53

  7. Dispersion suppressors with bending

    SciTech Connect

    Garren, A.

    1985-10-01

    Dispersion suppressors of two main types are usually used. In one the cell quadrupole focussing structure is the same as in normal cells but some of the dipoles are replaced by drifts. In the other, the quadrupole strengths and/or spacings are different from those of the normal cells, but the bending is about the same as it is in the cells. In SSC designs to date, dispersion suppressors of the former type have been used, consisting of two cells with bending equivalent to one. In this note a suppressor design with normal bending and altered focussing is presented. The advantage of this scheme is that circumference is reduced. The disadvantages are that additional special quadrupoles must be provided (however, they need not be adjustable), and the maximum beta values within them are about 30% higher than the cell maxima.

  8. Tumor suppressor identified as inhibitor of inflammation

    Cancer.gov

    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. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    PubMed

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  10. Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.

    PubMed

    N'guessan, Prudence; Pouyet, Laurent; Gosset, Gaëlle; Hamlaoui, Sonia; Seillier, Marion; Cano, Carla E; Seux, Mylène; Stocker, Pierre; Culcasi, Marcel; Iovanna, Juan L; Dusetti, Nelson J; Pietri, Sylvia; Carrier, Alice

    2011-09-15

    The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury.

  11. Syndecan-2 Exerts Antifibrotic Effects by Promoting Caveolin-1–mediated Transforming Growth Factor-β Receptor I Internalization and Inhibiting Transforming Growth Factor-β1 Signaling

    PubMed Central

    Shi, Yuanyuan; Gochuico, Bernadette R.; Yu, Guoying; Tang, Xiaomeng; Osorio, Juan C.; Fernandez, Isis E.; Risquez, Cristobal F.; Patel, Avignat S.; Shi, Ying; Wathelet, Marc G.; Goodwin, Andrew J.; Haspel, Jeffrey A.; Ryter, Stefan W.; Billings, Eric M.; Kaminski, Naftali; Morse, Danielle

    2013-01-01

    Rationale: Alveolar transforming growth factor (TGF)-β1 signaling and expression of TGF-β1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-β receptor TβRI inhibits TGF-β signaling and could attenuate development of experimental lung fibrosis. Objectives: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-β1 signaling in alveolar epithelial cells. Methods: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2–dependent changes in epithelial cell TGF-β1 signaling, TGF-β1, and TβRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. Measurements and Main Results: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-β1 signaling and downstream expression of TGF-β1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1–dependent internalization of TGF-β1 and TβRI in alveolar epithelial cells, which inhibited TGF-β1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. Conclusions: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1–dependent TGF-β1 and TβRI internalization and inhibiting TGF-β1 signaling in alveolar epithelial

  12. Myeloid derived suppressor cells

    PubMed Central

    Waldron, Todd J.; Quatromoni, Jon G.; Karakasheva, Tatiana A.; Singhal, Sunil; Rustgi, Anil K.

    2013-01-01

    The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy. PMID:23734336

  13. Sites in human nuclei where damage induced by ultraviolet light is repaired: localization relative to transcription sites and concentrations of proliferating cell nuclear antigen and the tumour suppressor protein, p53.

    PubMed

    Jackson, D A; Hassan, A B; Errington, R J; Cook, P R

    1994-07-01

    The repair of damage induced in DNA by ultraviolet light involves excision of the damaged sequence and synthesis of new DNA to repair the gap. Sites of such repair synthesis were visualized by incubating permeabilized HeLa or MRC-5 cells with the DNA precursor, biotin-dUTP, in a physiological buffer; then incorporated biotin was immunolabeled with fluorescent antibodies. Repair did not take place at sites that reflected the DNA distribution; rather, sites were focally concentrated in a complex pattern. This pattern changed with time; initially intense repair took place at transcriptionally active sites but when transcription became inhibited it continued at sites with little transcription. Repair synthesis in vitro also occurred in the absence of transcription. Repair sites generally contained a high concentration of proliferating cell nuclear antigen but not the tumour-suppressor protein, p53.

  14. Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway.

    PubMed

    Lv, Hongjun; Liu, Rui; Fu, Jiao; Yang, Qi; Shi, Jing; Chen, Pu; Ji, Meiju; Shi, Bingyin; Hou, Peng

    2014-01-01

    Periostin is usually considered as an oncogene in diverse human cancers, including breast, prostate, colon, esophagus, and pancreas cancers, whereas it acts as a tumor suppressor in bladder cancer. In gastric cancer, it has been demonstrated that periglandular periostin expression is decreased whereas stromal periostin expression is significantly increased as compared with normal gastric tissues. Moreover, periostin produced by stromal myofibroblasts markedly promotes gastric cancer cell growth. These observations suggest that periostin derived from different types of cells may play distinct biological roles in gastric tumorigenesis. The aim of this study was to explore the biological functions and related molecular mechanisms of epithelial cell-derived periostin in gastric cancer. Our data showed that periglandular periostin was significantly down-regulated in gastric cancer tissues as compared with matched normal gastric mucosa. In addition, its expression in metastatic lymph nodes was significantly lower than that in their primary cancer tissues. Our data also demonstrated that periglandular periostin expression was negatively associated with tumor stage. More importantly, restoration of periostin expression in gastric cancer cells dramatically suppressed cell growth and invasiveness. Elucidation of the mechanisms involved revealed that periostin restoration enhanced Rb phosphorylation and sequentially activated the transcription of E2F1 target gene p14(ARF), leading to Mdm2 inactivation and the stabilization of p53 and E-cadherin proteins. Strikingly, these effects of periostin were abolished upon Rb deletion. Collectively, we have for the first time demonstrated that epithelial cell-derived periostin exerts tumor-suppressor activities in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14(ARF)/Mdm2 signaling pathway. PMID:25486483

  15. Targeting tumor suppressor genes for cancer therapy.

    PubMed

    Liu, Yunhua; Hu, Xiaoxiao; Han, Cecil; Wang, Liana; Zhang, Xinna; He, Xiaoming; Lu, Xiongbin

    2015-12-01

    Cancer drugs are broadly classified into two categories: cytotoxic chemotherapies and targeted therapies that specifically modulate the activity of one or more proteins involved in cancer. Major advances have been achieved in targeted cancer therapies in the past few decades, which is ascribed to the increasing understanding of molecular mechanisms for cancer initiation and progression. Consequently, monoclonal antibodies and small molecules have been developed to interfere with a specific molecular oncogenic target. Targeting gain-of-function mutations, in general, has been productive. However, it has been a major challenge to use standard pharmacologic approaches to target loss-of-function mutations of tumor suppressor genes. Novel approaches, including synthetic lethality and collateral vulnerability screens, are now being developed to target gene defects in p53, PTEN, and BRCA1/2. Here, we review and summarize the recent findings in cancer genomics, drug development, and molecular cancer biology, which show promise in targeting tumor suppressors in cancer therapeutics.

  16. Caveolin-1 deficiency induces a MEK-ERK1/2-Snail-1-dependent epithelial–mesenchymal transition and fibrosis during peritoneal dialysis

    PubMed Central

    Strippoli, Raffaele; Loureiro, Jesús; Moreno, Vanessa; Benedicto, Ignacio; Pérez Lozano, María Luisa; Barreiro, Olga; Pellinen, Teijo; Minguet, Susana; Foronda, Miguel; Osteso, Maria Teresa; Calvo, Enrique; Vázquez, Jesús; López Cabrera, Manuel; del Pozo, Miguel Angel

    2015-01-01

    Peritoneal dialysis (PD) is a form of renal replacement therapy whose repeated use can alter dialytic function through induction of epithelial–mesenchymal transition (EMT) and fibrosis, eventually leading to PD discontinuation. The peritoneum from Cav1−/− mice showed increased EMT, thickness, and fibrosis. Exposure of Cav1−/− mice to PD fluids further increased peritoneal membrane thickness, altered permeability, and increased the number of FSP-1/cytokeratin-positive cells invading the sub-mesothelial stroma. High-throughput quantitative proteomics revealed increased abundance of collagens, FN, and laminin, as well as proteins related to TGF-β activity in matrices derived from Cav1−/− cells. Lack of Cav1 was associated with hyperactivation of a MEK-ERK1/2-Snail-1 pathway that regulated the Smad2-3/Smad1-5-8 balance. Pharmacological blockade of MEK rescued E-cadherin and ZO-1 inter-cellular junction localization, reduced fibrosis, and restored peritoneal function in Cav1−/− mice. Moreover, treatment of human PD-patient-derived MCs with drugs increasing Cav1 levels, as well as ectopic Cav1 expression, induced re-acquisition of epithelial features. This study demonstrates a pivotal role of Cav1 in the balance of epithelial versus mesenchymal state and suggests targets for the prevention of fibrosis during PD. PMID:25550395

  17. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    PubMed Central

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  18. Tumor suppressor protein Lgl mediates G1 cell cycle arrest at high cell density by forming an Lgl-VprBP-DDB1 complex

    PubMed Central

    Yamashita, Kazunari; Ide, Mariko; Furukawa, Kana T.; Suzuki, Atsushi; Hirano, Hisashi; Ohno, Shigeo

    2015-01-01

    Lethal giant larvae (Lgl) is an evolutionarily conserved tumor suppressor whose loss of function causes disrupted epithelial architecture with enhanced cell proliferation and defects in cell polarity. A role for Lgl in the establishment and maintenance of cell polarity via suppression of the PAR-aPKC polarity complex is established; however, the mechanism by which Lgl regulates cell proliferation is not fully understood. Here we show that depletion of Lgl1 and Lgl2 in MDCK epithelial cells results in overproliferation and overproduction of Lgl2 causes G1 arrest. We also show that Lgl associates with the VprBP-DDB1 complex independently of the PAR-aPKC complex and prevents the VprBP-DDB1 subunits from binding to Cul4A, a central component of the CRL4 [VprBP] ubiquitin E3 ligase complex implicated in G1- to S-phase progression. Consistently, depletion of VprBP or Cul4 rescues the overproliferation of Lgl-depleted cells. In addition, the affinity between Lgl2 and the VprBP-DDB1 complex increases at high cell density. Further, aPKC-mediated phosphorylation of Lgl2 negatively regulates the interaction between Lgl2 and VprBP-DDB1 complex. These results suggest a mechanism protecting overproliferation of epithelial cells in which Lgl plays a critical role by inhibiting formation of the CRL4 [VprBP] complex, resulting in G1 arrest. PMID:25947136

  19. Toll-Like Receptor 3 and Suppressor of Cytokine Signaling Proteins Regulate CXCR4 and CXCR7 Expression in Bone Marrow-Derived Human Multipotent Stromal Cells

    PubMed Central

    Tomchuck, Suzanne L.; Henkle, Sarah L.; Coffelt, Seth B.; Betancourt, Aline M.

    2012-01-01

    Background The use of bone marrow-derived human multipotent stromal cells (hMSC) in cell-based therapies has dramatically increased in recent years, as researchers have exploited the ability of these cells to migrate to sites of tissue injury, inflammation, and tumors. Our group established that hMSC respond to “danger” signals – by-products of damaged, infected or inflamed tissues – via activation of Toll-like receptors (TLRs). However, little is known regarding downstream signaling mediated by TLRs in hMSC. Methodology/Principal Findings We demonstrate that TLR3 stimulation activates a Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 1 pathway, and increases expression of suppressor of cytokine signaling (SOCS) 1 and SOCS3 in hMSC. Our studies suggest that each of these SOCS plays a distinct role in negatively regulating TLR3 and JAK/STAT signaling. TLR3-mediated interferon regulatory factor 1 (IRF1) expression was inhibited by SOCS3 overexpression in hMSC while SOCS1 overexpression reduced STAT1 activation. Furthermore, our study is the first to demonstrate that when TLR3 is activated in hMSC, expression of CXCR4 and CXCR7 is downregulated. SOCS3 overexpression inhibited internalization of both CXCR4 and CXCR7 following TLR3 stimulation. In contrast, SOCS1 overexpression only inhibited CXCR7 internalization. Conclusion/Significance These results demonstrate that SOCS1 and SOCS3 each play a functionally distinct role in modulating TLR3, JAK/STAT, and CXCR4/CXCR7 signaling in hMSC and shed further light on the way hMSC respond to danger signals. PMID:22745793

  20. Runt-related Transcription Factor 1 (RUNX1) Stimulates Tumor Suppressor p53 Protein in Response to DNA Damage through Complex Formation and Acetylation*

    PubMed Central

    Wu, Dan; Ozaki, Toshinori; Yoshihara, Yukari; Kubo, Natsumi; Nakagawara, Akira

    2013-01-01

    Representative tumor suppressor p53 plays a critical role in the regulation of proper DNA damage response. In this study, we have found for the first time that Runt-related transcription factor 1 (RUNX1) contributes to p53-dependent DNA damage response. Upon adriamycin (ADR) exposure, p53 as well as RUNX1 were strongly induced in p53-proficient HCT116 and U2OS cells, which were closely associated with significant transactivation of p53 target genes, such as p21WAF1, BAX, NOXA, and PUMA. RUNX1 was exclusively expressed in the cell nucleus and formed a complex with p53 in response to ADR. Chromatin immunoprecipitation assay demonstrated that p53 together with RUNX1 are efficiently recruited onto p53 target gene promoters following ADR exposure, indicating that RUNX1 is involved in p53-mediated transcriptional regulation. Indeed, forced expression of RUNX1 stimulated the transcriptional activity of p53 in response to ADR. Consistent with these observations, knockdown of RUNX1 attenuated ADR-mediated induction of p53 target genes and suppressed ADR-dependent apoptosis. Furthermore, RUNX1 was associated with p300 histone acetyltransferase, and ADR-dependent acetylation of p53 at Lys-373/382 was markedly inhibited in RUNX1 knockdown cells. In addition, knockdown of RUNX1 resulted in a significant decrease in the amount of p53-p300 complex following ADR exposure. Taken together, our present results strongly suggest that RUNX1 is required for the stimulation of p53 in response to DNA damage and also provide novel insight into understanding the molecular mechanisms behind p53-dependent DNA damage response. PMID:23148227

  1. Tumor suppressor molecules and methods of use

    DOEpatents

    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.

  2. The Role of Suppressors of Cytokine Signalling in Human Neoplasms

    PubMed Central

    Sharma, Anup K.; Mokbel, Kefah

    2014-01-01

    Suppressors of cytokine signalling 1–7 (SOCS1–7) and cytokine-inducible SH2-containing protein (CIS) are a group of intracellular proteins that are well known as JAK-STAT and several other signalling pathways negative feedback regulators. More recently several members have been identified as tumour suppressors and dysregulation of their biological roles in controlling cytokine and growth factor signalling may contribute to the development of many solid organ and haematological malignancies. This review explores their biological functions and their possible tumour suppressing role in human neoplasms. PMID:24757565

  3. The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

    PubMed Central

    Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

    2012-01-01

    The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

  4. Vaccinia virus A25 and A26 proteins are fusion suppressors for mature virions and determine strain-specific virus entry pathways into HeLa, CHO-K1, and L cells.

    PubMed

    Chang, Shu-Jung; Chang, Yu-Xun; Izmailyan, Roza; Tang, Yin-Liang; Chang, Wen

    2010-09-01

    Mature vaccinia virus enters cells through either fluid-phase endocytosis/macropinocytosis or plasma membrane fusion. This may explain the wide range of host cell susceptibilities to vaccinia virus entry; however, it is not known how vaccinia virus chooses between these two pathways and which viral envelope proteins determine such processes. By screening several recombinant viruses and different strains, we found that mature virions containing the vaccinia virus A25 and A26 proteins entered HeLa cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two proteins entered through a bafilomycin-resistant pathway. To investigate whether the A25 and A26 proteins contribute to entry pathway specificity, two mutant vaccinia viruses, WRDeltaA25L and WRDeltaA26L, were subsequently generated from the wild-type WR strain. In contrast to the WR strain, both the WRDeltaA25L and WRDeltaA26L viruses became resistant to bafilomycin, suggesting that the removal of the A25 and A26 proteins bypassed the low-pH endosomal requirement for mature virion entry. Indeed, WRDeltaA25L and WRDeltaA26L virus infections of HeLa, CHO-K1, and L cells immediately triggered cell-to-cell fusion at a neutral pH at 1 to 2 h postinfection (p.i.), providing direct evidence that viral fusion machinery is readily activated after the removal of the A25 and A26 proteins to allow virus entry through the plasma membrane. In summary, our data support a model that on vaccinia mature virions, the viral A25 and A26 proteins are low-pH-sensitive fusion suppressors whose inactivation during the endocytic route results in viral and cell membrane fusion. Our results also suggest that during virion morphogenesis, the incorporation of the A25 and A26 proteins into mature virions may help restrain viral fusion activity until the time of infections.

  5. Suppressor of hairy‐wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi

    PubMed Central

    Carballar‐Lejarazú, R.; Brennock, P.

    2016-01-01

    Abstract DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy‐like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric‐a‐brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood‐fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes. PMID:27110891

  6. Suppressor of hairy-wing, modifier of mdg4 and centrosomal protein of 190 gene orthologues of the gypsy insulator complex in the malaria mosquito, Anopheles stephensi.

    PubMed

    Carballar-Lejarazú, R; Brennock, P; James, A A

    2016-08-01

    DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.

  7. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

    PubMed Central

    Craven, R J; Petes, T D

    2001-01-01

    Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p. PMID:11333225

  8. Tumor suppressor von Hippel-Lindau (VHL) stabilization of Jade-1 protein occurs through plant homeodomains and is VHL mutation dependent.

    PubMed

    Zhou, Mina I; Wang, Hongmei; Foy, Rebecca L; Ross, Jonathan J; Cohen, Herbert T

    2004-02-15

    The von Hippel-Lindau (VHL) gene is the major renal cancer gene in adults. The mechanism of renal tumor suppression by VHL protein is only partly elucidated. VHL loss increases expression of the hypoxia-inducible factor alpha transcription factors. However, clinical and biochemical data indicate that the hypoxia-inducible factors are necessary but not sufficient for renal tumorigenesis, which suggests other VHL effector pathways are involved. Jade-1 protein interacts strongly with VHL and is most highly expressed in renal proximal tubules, precursor cells of renal cancer. Short-lived Jade-1 protein contains plant homeodomain (PHD) and candidate PEST degradation motifs and is substantially stabilized by VHL. The effect of VHL on Jade-1 protein abundance and relative protein stability was further examined in immunoblots and metabolic labeling experiments using two time points. VHL-Jade-1 binding was tested in coimmunoprecipitations. In cotransfection studies with wild-type VHL, the Jade-1 PHD-extended PHD module, not the candidate PEST domain, was required for full VHL-mediated stabilization. This module is also found in leukemia transcription factors AF10 and AF17, as well as closely related Jade-like proteins, which suggests all might be VHL regulated. Intriguingly, naturally occurring truncations and mutations of VHL affected wild-type Jade-1 binding and stabilization. Although the VHL beta domain was sufficient for Jade-1 binding, both the alpha and beta domains were required for Jade-1 stabilization. Thus, truncating VHL mutations, which are severe and associated with renal cancer development, prevented Jade-1 stabilization. Moreover, well-controlled cotransfection and metabolic labeling experiments revealed that VHL missense mutations that cause VHL disease without renal cancer, such as Tyr98His and Tyr112His, stabilized Jade-1 fully. In contrast, like the VHL truncations, VHL missense mutations commonly associated with renal cancer, such as Leu118Pro or Arg167

  9. Endoproteolytic cleavage of FE65 converts the adaptor protein to a potent suppressor of the sAPPalpha pathway in primates.

    PubMed

    Hu, Qubai; Wang, Lin; Yang, Zheng; Cool, Bethany H; Zitnik, Galynn; Martin, George M

    2005-04-01

    Adaptor protein FE65 (APBB1) specifically binds to the intracellular tail of the type I transmembrane protein, beta-amyloid precursor protein (APP). The formation of this complex may be important for modulation of the processing and function of APP. APP is proteolytically cleaved at multiple sites. The cleavages and their regulation are of central importance in the pathogenesis of dementias of the Alzheimer type. In cell cultures and perhaps in vivo, secretion of the alpha-cleaved APP ectodomain (sAPPalpha) is the major pathway in the most cells. Regulation of the process may require extracellular/intracellular cues. Neither extracellular ligands nor intracellular mediators have been identified, however. Here, we show novel evidence that the major isoform of FE65 (97-kDa FE65, p97FE65) can be converted to a 65-kDa N-terminally truncated C-terminal fragment (p65FE65) via endoproteolysis. The cleavage region locates immediately after an acidic residue cluster but before the three major protein-protein binding domains. The cleavage activity is particularly high in human and non-human primate cells and low in rodent cells; the activity appears to be triggered/enhanced by high cell density, presumably via cell-cell/cell-substrate contact cues. As a result, p65FE65 exhibits extraordinarily high affinity for APP (up to 40-fold higher than p97FE65) and potent suppression (up to 90%) of secretion of sAPPalpha. Strong p65FE65-APP binding is required for the suppression. The results suggest that p65FE65 may be an intracellular mediator in a signaling cascade regulating alpha-secretion of APP, particularly in primates. PMID:15647266

  10. Tumor Suppressor Analysis in CML.

    PubMed

    Herrmann, Oliver; Schemionek, Mirle

    2016-01-01

    Retroviral models have tremendously contributed to our understanding of CML development and have been indispensable for preclinical drug testing which facilitated the implementation of a targeted therapy. The retroviral insertion of Bcr-Abl into mice that are genetically depleted for a potential tumor suppressor is a tool to test for a specific gene function in Bcr-Abl disease. Here we describe how to generate a Bcr-Abl retrovirus that is subsequently used for infection of primary murine BM cells, which are genetically depleted for a potential tumor suppressor gene. We will suggest control experiments and outline further methods that are required to allow for assessment of disease development upon tumor suppressor knockout in CML. PMID:27581141

  11. Membrane Association of the PTEN Tumor Suppressor: Molecular Details of the Protein-Membrane Complex from SPR Binding Studies and Neutron Reflection

    PubMed Central

    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 <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the 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

  12. 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.

    PubMed

    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. PMID:26244501

  13. 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.

    PubMed

    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.

  14. The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNA(GLN) (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation.

    PubMed

    Hohmann, S; Van Dijck, P; Luyten, K; Thevelein, J M

    1994-10-01

    Byp1-3 is an amber nonsense allele of the Saccharomyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 delta mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 delta mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA(GLN) (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA(GLN) (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA(GLN) (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA(GLN) (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.

  15. Shear-stress-mediated refolding of proteins from aggregates and inclusion bodies.

    PubMed

    Yuan, Tom Z; Ormonde, Callum F G; Kudlacek, Stephan T; Kunche, Sameeran; Smith, Joshua N; Brown, William A; Pugliese, Kaitlin M; Olsen, Tivoli J; Iftikhar, Mariam; Raston, Colin L; Weiss, Gregory A

    2015-02-01

    Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.

  16. Characterization of DNA Binding Property of the HIV-1 Host Factor and Tumor Suppressor Protein Integrase Interactor 1 (INI1/hSNF5)

    PubMed Central

    Das, Supratik; Thangamuniyandi, Muruganandan; Dasgupta, Saumya; Chongdar, Nipa; Kumar, Gopinatha Suresh; Basu, Gautam

    2013-01-01

    Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105–183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K’d1 = 222 µM and K’d2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = –29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH’1 = 115.7 KJ/mole, TΔS’1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH’2 = –106.3 KJ/mole, TΔS’2 = –75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1. PMID:23861745

  17. The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition.

    PubMed

    Albert, Thomas K; Antrecht, Claudia; Kremmer, Elisabeth; Meisterernst, Michael

    2016-01-01

    CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription. PMID:26745862

  18. Active oxygen species mediate the solar ultraviolet radiation-dependent increase in the tumour suppressor protein p53 in human skin fibroblasts.

    PubMed

    Vile, G F

    1997-07-21

    Active oxygen species mediate many of the biological consequences of exposing cultured human skin cells to solar ultraviolet (UV) radiation (290-380 nm). A critical step in the escape from the carcinogenic potential of UV radiation is mediated by the protein p53. P53 activates growth arrest, allowing for DNA repair, and apoptosis, which removes damaged cells. Here I show that p53 in cultured human skin fibroblasts is elevated after treatment with hydrogen peroxide, an oxidant produced in cells during exposure to solar UV radiation. Simulated solar UV radiation increased p53, and agents that scavenge active oxygen species, N-acetylcysteine, ascorbate and alpha-tocopherol, inhibited the increase. The generation of DNA single strand breaks has been proposed to be an important step in the pathway leading to the increase in p53 initiated by a variety of cytotoxic agents. In this study I show that compounds that allow the accumulation of DNA single strand breaks, ara c and hydroxyurea, enhanced the UVC radiation (254 nm)-dependent increase in p53, but had no effect on the solar UV radiation-dependent increase. Thus, while DNA single strand breaks are involved in the UVC radiation-dependent increase in p53, the increase caused by solar UV radiation occurs by an alternative mechanism involving active oxygen species.

  19. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin.

    PubMed

    Hoy, Benjamin; Geppert, Tim; Boehm, Manja; Reisen, Felix; Plattner, Patrick; Gadermaier, Gabriele; Sewald, Norbert; Ferreira, Fatima; Briza, Peter; Schneider, Gisbert; Backert, Steffen; Wessler, Silja

    2012-03-23

    The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections. PMID:22337879

  20. New insight on the biological role of p53 protein as a tumor suppressor: re-evaluation of its clinical significance in triple-negative breast cancer.

    PubMed

    Jin, Min-Sun; Park, In Ae; Kim, Ji Young; Chung, Yul Ri; Im, Seock-Ah; Lee, Kyung-Hun; Moon, Hyeong-Gon; Han, Wonshik; Noh, Dong-Young; Ryu, Han Suk

    2016-08-01

    While p53 mutation is found in the majority of triple-negative breast cancer (TNBC) and despite recent developments in p53-targeting agents, their therapeutic application is still limited by the absence of standard biomarkers and ambiguousness of its essential biological role in cancer. Whole sections from 305 TNBC cases were stained for p53 to determine the correlation with lymph node metastasis and clinical outcomes in the whole cohort as well as in stratified patient groups according to AJCC stage and the use of adjuvant chemotherapy. Reduced immunohistochemical expression of p53 was an independent risk factor for lymph node metastasis. p53 overexpression was predictive of better clinical outcome in all patients (P = 0.012, disease-free survival and P = 0.008, overall survival) and the stratified cohorts of those who had early breast cancer and received adjuvant chemotherapy. Suppression of endogenous mutant p53 by siRNA and induction of wild-type p53 repressed TNBC cell invasion in vitro. In TNBC, increased immunohistochemical expression of p53 may reflect the accumulation of wild-type p53 rather than the mutant form. Strong p53 protein expression may serve as a favorable prognostic indicator and provide evidence for the use of specific agents targeting p53.

  1. miRNA-411 acts as a potential tumor suppressor miRNA via the downregulation of specificity protein 1 in breast cancer

    PubMed Central

    Guo, Liangfeng; Yuan, Jianhui; Xie, Ni; Wu, Huisheng; Chen, Weicai; Song, Shufen; Wang, Xianming

    2016-01-01

    The expression and functions of microRNA (miR)-411 have been investigated in several types of cancer. However, until now, miR-411 in human breast cancer has not been examined. The present study investigated the expression, biological functions and molecular mechanisms of miR-411 in human breast cancer, discussing whether it offers potential as a therapeutic biomarker for breast cancer in the future. The expression levels of miR-411 in human breast cancer tissues and cells were measured using reverse transcription-quantitative polymerase chain reaction analysis. Following transfection with miR-411 mimics, an MTT assay, cell migration and invasion assay, western blot analysis and luciferase assay were performed in human breast cancer cell lines. According to the results, it was found that miR-411 was significantly downregulated in breast cancer, and associated with lymph node metastasis and histological grade. Additionally, it was observed that miR-411 suppressed cell growth, migration and invasion in the breast cancer cells. The present study also provided the first evidence, to the best of our knowledge, that miR-411 was likely to directly target specificity protein 1 in breast cancer. These findings indicated that miR-411 may be used a therapeutic biomarker for the treatment of breast cancer in the future. PMID:27572271

  2. The extreme COOH terminus of the retinoblastoma tumor suppressor protein pRb is required for phosphorylation on Thr-373 and activation of E2F.

    PubMed

    Gorges, Laura L; Lents, Nathan H; Baldassare, Joseph J

    2008-11-01

    The retinoblastoma protein pRb plays a pivotal role in G(1)- to S-phase cell cycle progression and is among the most frequently mutated gene products in human cancer. Although much focus has been placed on understanding how the A/B pocket and COOH-terminal domain of pRb cooperate to relieve transcriptional repression of E2F-responsive genes, comparatively little emphasis has been placed on the function of the NH(2)-terminal region of pRb and the interaction of the multiple domains of pRb in the full-length context. Using "reverse mutational analysis" of Rb(DeltaCDK) (a dominantly active repressive allele of Rb), we have previously shown that restoration of Thr-373 is sufficient to render Rb(DeltaCDK) sensitive to inactivation via cyclin-CDK phosphorylation. This suggests that the NH(2)-terminal region plays a more critical role in pRb regulation than previously thought. In the present study, we have expanded this analysis to include additional residues in the NH(2)-terminal region of pRb and further establish that the mechanism of pRb inactivation by Thr-373 phosphorylation is through the dissociation of E2F. Most surprisingly, we further have found that removal of the COOH-terminal domain of either RbDeltaCDK(+T373) or wild-type pRb yields a functional allele that cannot be inactivated by phosphorylation and is repressive of E2F activation and S-phase entry. Our data demonstrate a novel function for the NH(2)-terminal domain of pRb and the necessity for cooperation of multiple domains for proper pRb regulation.

  3. Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles

    PubMed Central

    He, Jian; Zheng, Yi-Wen; Lin, Yi-Fan; Mi, Shu; Qin, Xiao-Wei; Weng, Shao-Ping; He, Jian-Guo; Guo, Chang-Jun

    2016-01-01

    Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-β-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions. PMID:26887868

  4. Caveolae Restrict Tiger Frog Virus Release in HepG2 cells and Caveolae-Associated Proteins Incorporated into Virus Particles.

    PubMed

    He, Jian; Zheng, Yi-Wen; Lin, Yi-Fan; Mi, Shu; Qin, Xiao-Wei; Weng, Shao-Ping; He, Jian-Guo; Guo, Chang-Jun

    2016-01-01

    Caveolae are flask-shaped invaginations of the plasma membrane. Caveolae play important roles in the process of viruses entry into host cells, but the roles of caveolae at the late stage of virus infection were not completely understood. Tiger frog virus (TFV) has been isolated from the diseased tadpoles of the frog, Rana tigrina rugulosa, and causes high mortality of tiger frog tadpoles cultured in Southern China. In the present study, the roles of caveolae at the late stage of TFV infection were investigated. We showed that TFV virions were localized with the caveolae at the late stage of infection in HepG2 cells. Disruption of caveolae by methyl-β-cyclodextrin/nystatin or knockdown of caveolin-1 significantly increase the release of TFV. Moreover, the interaction between caveolin-1 and TFV major capsid protein was detected by co-immunoprecipitation. Those results suggested that caveolae restricted TFV release from the HepG2 cells. Caveolae-associated proteins (caveolin-1, caveolin-2, cavin-1, and cavin-2) were selectively incorporated into TFV virions. Different combinations of proteolytic and/or detergent treatments with virions showed that caveolae-associated proteins were located in viral capsid of TFV virons. Taken together, caveolae might be a restriction factor that affects virus release and caveolae-associated proteins were incorporated in TFV virions.

  5. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    SciTech Connect

    Kumari, Gita; Mahalingam, S.

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  6. Parenteral versus enteral nutrition: effect on serum cytokines and the hepatic expression of mRNA of suppressor of cytokine signaling proteins, insulin-like growth factor-1 and the growth hormone receptor in rodent sepsis

    PubMed Central

    O'Leary, Michael J; Xue, Aiqun; Scarlett, Christopher J; Sevette, Andre; Kee, Anthony J; Smith, Ross C

    2007-01-01

    Introduction Early nutrition is recommended for patients with sepsis, but data are conflicting regarding the optimum route of delivery. Enteral nutrition (EN), compared with parenteral nutrition (PN), results in poorer achievement of nutritional goals but may be associated with fewer infections. Mechanisms underlying differential effects of the feeding route on patient outcomes are not understood, but probably involve the immune system and the anabolic response to nutrients. We studied the effect of nutrition and the route of delivery of nutrition on cytokine profiles, the growth hormone–insulin-like growth factor-1 (IGF-I) axis and a potential mechanism for immune and anabolic system interaction, the suppressors of cytokine signaling (SOCS), in rodents with and without sepsis. Methods Male Sprague–Dawley rats were randomized to laparotomy (Sham) or to cecal ligation and puncture (CLP), with postoperative saline infusion (Starve), with EN or with PN for 72 hours. Serum levels of IL-6 and IL-10 were measured by immunoassay, and hepatic expressions of cytokine-inducible SH2-containing protein, SOCS-2, SOCS-3, IGF-I and the growth hormone receptor (GHR) were measured by real-time quantitative PCR. Results IL-6 was detectable in all groups, but was only present in all animals receiving CLP-PN. IL-10 was detectable in all but one CLP-PN rat, one CLP-EN rat, approximately 50% of the CLP-Starve rats and no sham-operated rats. Cytokine-inducible SH2-containing protein mRNA was increased in the CLP-EN group compared with the Sham-EN group and the other CLP groups (P < 0.05). SOCS-2 mRNA was decreased in CLP-PN rats compared with Sham-PN rats (P = 0.07). SOCS-3 mRNA was increased with CLP compared with sham operation (P < 0.03). IGF-I mRNA (P < 0.05) and GHR mRNA (P < 0.03) were greater in the fed CLP animals and in the Sham-PN group compared with the starved rats. Conclusion In established sepsis, nutrition and the route of administration of nutrition influences the

  7. Therapeutic Targeting of Tumor Suppressor Genes

    PubMed Central

    Morris, Luc G. T.; Chan, Timothy A.

    2015-01-01

    Carcinogenesis is a multistep process attributable to both gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes. Currently, most molecular targeted therapies are inhibitors of oncogenes, because inactivated tumor suppressor genes have proven harder to “drug.” Nevertheless, in cancers, tumor suppressor genes undergo alteration more frequently than do oncogenes. In recent years, several promising strategies directed at tumor suppressor genes, or the pathways controlled by these genes, have emerged. Here, we describe advances in a number of different methodologies aimed at therapeutically targeting tumors driven by inactivated tumor suppressor genes. PMID:25557041

  8. Electrodialytic membrane suppressors for ion chromatography make programmable buffer generators.

    PubMed

    Chen, Yongjing; Srinivasan, Kannan; Dasgupta, Purnendu K

    2012-01-01

    The use of buffer solutions is immensely important in a great variety of disciplines. The generation of continuous pH gradients in flow systems plays an important role in the chromatographic separation of proteins, high-throughput pK(a) determinations, etc. We demonstrate here that electrodialytic membrane suppressors used in ion chromatography can be used to generate buffers. The generated pH, computed from first principles, agrees well with measured values. We demonstrate the generation of phosphate and citrate buffers using a cation-exchange membrane (CEM) -based anion suppressor and Tris and ethylenediamine buffers using an anion-exchange membrane (AEM) -based cation suppressor. Using a mixture of phosphate, citrate, and borate as the buffering ions and using a CEM suppressor, we demonstrate the generation of a highly reproducible (avg RSD 0.20%, n = 3), temporally linear (pH 3.0-11.9, r(2) > 0.9996), electrically controlled pH gradient. With butylamine and a large concentration (0.5 M) of added NaCl, we demonstrate a similar linear pH gradient of large range with a near-constant ionic strength. We believe that this approach will be of value for the generation of eluents in the separation of proteins and other biomolecules and in online process titrations.

  9. Fms-Like Tyrosine Kinase 3 Ligand Decreases T Helper Type 17 Cells and Suppressors of Cytokine Signaling Proteins in the Lung of House Dust Mite–Sensitized and –Challenged Mice

    PubMed Central

    McGee, Halvor S.; Stallworth, Arthur L.; Agrawal, Tanupriya; Shao, Zhifei; Lorence, Lindsey; Agrawal, Devendra K.

    2010-01-01

    We previously reported that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11chighCD8αhighCD11blow dendritic cells and CD4+CD25+ICOS+Foxp3+IL-10+ T-regulatory cells in the lung of allergen-sensitized and -challenged mice. In this study, we evaluated the effect of Flt3-L on Th17 cells and expression of suppressors of cytokine signaling (SOCS) proteins in the lungs of house dust mite (HDM)–sensitized and –challenged mice. BALB/c mice were sensitized and challenged with HDM, and AHR to methacholine was established. Mice were treated with Flt3-L (5 μg, intraperitoneal) daily for 10 days. Levels of IL-4, -5, -6, -8, and -13, and transforming growth factor (TGF)–β in the bronchoalveolar lavage fluid (BALF) were examined by ELISA. Flt3-L treatment reversed existing AHR to methacholine and substantially decreased eosinophils, neutrophils, IL-5, -6, -8, and IL-13, and TGF-β levels in the BALF. HDM-sensitized and -challenged mice showed a significant increase in lung CD4+IL-17+IL-23R+CD25− T cells with high expression of retinoic acid–related orphan receptor (ROR)–γt transcripts. However, administration of Flt3-L substantially decreased the number of lung CD4+IL-17+IL-23R+CD25− T cells, with significantly decreased expression of ROR-γt mRNA in these cells. HDM sensitization caused a significant increase in the expression of SOCS-1, -3, and -5 in the lung. Flt3-L treatment abolished the increase in SOCS-1 and SOCS-3 proteins, whereas SOCS-5 expression was significantly reduced. These data suggest that the therapeutic effect of Flt3-L in reversing the hallmarks of allergic asthma in a mouse model is mediated by decreasing IL-6 and TGF-β levels in the BALF, which, in turn, decrease CD4+IL-17+IL-23R+ROR-γt+CD25− T cells and the expression of SOCS-1 and SOCS-3 in the lung of HDM-sensitized and -challenged mice. PMID:19933379

  10. Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

    PubMed Central

    van Mierlo, Joël T.; Overheul, Gijs J.; Obadia, Benjamin; van Cleef, Koen W. R.; Webster, Claire L.; Saleh, Maria-Carla; Obbard, Darren J.; van Rij, Ronald P.

    2014-01-01

    The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors. PMID:25032815

  11. Nuclear PTEN tumor-suppressor functions through maintaining heterochromatin structure.

    PubMed

    Gong, Lili; Govan, Jeane M; Evans, Elizabeth B; Dai, Hui; Wang, Edward; Lee, Szu-Wei; Lin, Hui-Kuan; Lazar, Alexander J; Mills, Gordon B; Lin, Shiaw-Yih

    2015-01-01

    The tumor suppressor, PTEN, is one of the most commonly mutated genes in cancer. Recently, PTEN has been shown to localize in the nucleus and is required to maintain genomic stability. Here, we show that nuclear PTEN, independent of its phosphatase activity, is essential for maintaining heterochromatin structure. Depletion of PTEN leads to loss of heterochromatic foci, decreased chromatin compaction, overexpression of heterochromatic genes, and reduced protein stability of heterochromatin protein 1 α. We found that the C-terminus of PTEN is required to maintain heterochromatin structure. Additionally, cancer-associated PTEN mutants lost their tumor-suppressor function when their heterochromatin structure was compromised. We propose that this novel role of PTEN accounts for its function in guarding genomic stability and suppressing tumor development. PMID:25946202

  12. Nuclear PTEN tumor-suppressor functions through maintaining heterochromatin structure

    PubMed Central

    Gong, Lili; Govan, Jeane M; Evans, Elizabeth B; Dai, Hui; Wang, Edward; Lee, Szu-Wei; Lin, Hui-Kuan; Lazar, Alexander J; Mills, Gordon B; Lin, Shiaw-Yih

    2015-01-01

    The tumor suppressor, PTEN, is one of the most commonly mutated genes in cancer. Recently, PTEN has been shown to localize in the nucleus and is required to maintain genomic stability. Here, we show that nuclear PTEN, independent of its phosphatase activity, is essential for maintaining heterochromatin structure. Depletion of PTEN leads to loss of heterochromatic foci, decreased chromatin compaction, overexpression of heterochromatic genes, and reduced protein stability of heterochromatin protein 1 α. We found that the C-terminus of PTEN is required to maintain heterochromatin structure. Additionally, cancer-associated PTEN mutants lost their tumor-suppressor function when their heterochromatin structure was compromised. We propose that this novel role of PTEN accounts for its function in guarding genomic stability and suppressing tumor development. PMID:25946202

  13. Noise suppressor for turbo fan jet engines

    NASA Technical Reports Server (NTRS)

    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.

  14. Suppressors made from intermetallic materials

    SciTech Connect

    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. Identification of phosphatidylcholine transfer protein-like in the parasite Entamoeba histolytica.

    PubMed

    Piña-Vázquez, Carolina; Reyes-López, Magda; Mendoza-Hernández, Guillermo; Bermúdez-Cruz, Rosa María; de la Garza, Mireya

    2014-12-01

    Caveolin is the protein marker of caveola-mediated endocytosis. Previously, we demonstrated by immunoblotting and immunofluorescence that an anti-chick embryo caveolin-1 monoclonal antibody (mAb) recognizes a protein in amoeba extracts. Nevertheless, the caveolin-1 gene is absent in the Entamoeba histolytica genome database. In this work, the goal was to isolate, identify and characterize the protein that cross-reacts with chick embryo caveolin-1. We identified the protein using a proteomic approach, and the complete gene was cloned and sequenced. The identified protein, E. histolytica phosphatidylcholine transfer protein-like (EhPCTP-L), is a member of the StAR-related lipid transfer (START) protein superfamily. The human homolog binds and transfers phosphatidylcholine (PC) and phosphatidylethanolamine (PE) between model membranes in vitro; however, the physiological role of PCTP-L remains elusive. Studies in silico showed that EhPCTP-L has a central START domain and also contains a C-terminal intrinsically disordered region. The anti-rEhPCTP-L antibody demonstrated that EhPCTP-L is found in the plasma membrane and cytosol, which is in agreement with previous reports on the human counterpart. This result points to the plasma membrane as one possible target membrane for EhPCTP-L. Furthermore, assays using filipin and nystatin showed down regulation of EhPCTP-L, in an apparently cholesterol-independent way. Interestingly, EhPCTP-L binds primarily to anionic phospholipids phosphatidylserine (PS) and phosphatidic acid (PA), while its mammalian counterpart HsPCTP-L binds neutral phospholipids PC and PE. The present study provides information that helps reveal the possible function and regulation of PCTP-L expression in the primitive eukaryotic parasite E. histolytica.

  16. Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants.

    PubMed

    Hemmes, Hans; Kaaij, Lucas; Lohuis, Dick; Prins, Marcel; Goldbach, Rob; Schnettler, Esther

    2009-07-01

    The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at positions 173-175 are involved in the siRNA binding and suppressor activities. PMID:19282433

  17. Suppressor Effects of Coping Strategies on Resilience

    ERIC Educational Resources Information Center

    Yoon, Jae ho; Lee, Ji hae; Lee, Chae Yeon; Cho, Minhee; Lee, Sang Min

    2014-01-01

    The purpose of the current study is to demonstrate a significant suppressor effect among coping strategies on resilience. Two different samples were used to replicate the suppressor effect. Participants in the first example were 391 adolescents (middle school students) in Korea, and participants in the second example were 282 young adults…

  18. Jet mixer noise suppressor using acoustic feedback

    NASA Technical Reports Server (NTRS)

    Rice, Edward J. (Inventor)

    1995-01-01

    The present invention generally relates to providing an improved jet mixer noise suppressor for high speed jets that rapidly mixes high speed air flow with a lower speed air flow, and more particularly, relates to an improved jet mixer noise suppressor that uses feedback of acoustic waves produced by the interaction of shear flow instability waves with an obstacle downstream of the jet nozzle.

  19. Jet mixer noise suppressor using acoustic feedback

    NASA Technical Reports Server (NTRS)

    Rice, Edward J. (Inventor)

    1994-01-01

    The present invention generally relates to providing an improved jet mixer noise suppressor for high speed jets that rapidly mixes high speed air flow with a lower speed air flow, and more particularly, relates to an improved jet mixer noise suppressor that uses feedback of acoustic waves produced by the interaction of sheer flow instability waves with an obstacle downstream of the jet nozzle.

  20. Discovery of Tumor Suppressor Gene Function.

    ERIC Educational Resources Information Center

    Oppenheimer, Steven B.

    1995-01-01

    This is an update of a 1991 review on tumor suppressor genes written at a time when understanding of how the genes work was limited. A recent major breakthrough in the understanding of the function of tumor suppressor genes is discussed. (LZ)

  1. Suppressors of RNA silencing encoded by tomato leaf curl betasatellites.

    PubMed

    Shukla, Richa; Dalal, Sunita; Malathi, V G

    2013-03-01

    Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. 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 and ORF beta C1 in satellite DNA beta which are predicted to function as silencing suppressors. In the present study suppressor function of ORF beta C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB-[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing betaC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with betaC1 gene from ToLCBB. However, in the case of 35S-beta C1 CLCuMB and 35S- beta C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which beta C1 interfere in RNA-silencing pathway is different. PMID:23385812

  2. PML, a growth suppressor disrupted in acute promyelocytic leukemia.

    PubMed Central

    Mu, Z M; Chin, K V; Liu, J H; Lozano, G; Chang, K S

    1994-01-01

    The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis. Images PMID:7935403

  3. Targeting tumor suppressor networks for cancer therapeutics.

    PubMed

    Guo, Xuning Emily; Ngo, Bryan; Modrek, Aram Sandaldjian; Lee, Wen-Hwa

    2014-01-01

    Cancer is a consequence of mutations in genes that control cell proliferation, differentiation and cellular homeostasis. These genes are classified into two categories: oncogenes and tumor suppressor genes. Together, overexpression of oncogenes and loss of tumor suppressors are the dominant driving forces for tumorigenesis. Hence, targeting oncogenes and tumor suppressors hold tremendous therapeutic potential for cancer treatment. In the last decade, the predominant cancer drug discovery strategy has relied on a traditional reductionist approach of dissecting molecular signaling pathways and designing inhibitors for the selected oncogenic targets. Remarkable therapies have been developed using this approach; however, targeting oncogenes is only part of the picture. Our understanding of the importance of tumor suppressors in preventing tumorigenesis has also advanced significantly and provides a new therapeutic window of opportunity. Given that tumor suppressors are frequently mutated, deleted, or silenced with loss-of-function, restoring their normal functions to treat cancer holds tremendous therapeutic potential. With the rapid expansion in our knowledge of cancer over the last several decades, developing effective anticancer regimens against tumor suppressor pathways has never been more promising. In this article, we will review the concept of tumor suppression, and outline the major therapeutic strategies and challenges of targeting tumor suppressor networks for cancer therapeutics.

  4. Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a new member of the Cupin superfamily. A possible multifunctional protein acting as an invasion suppressor down-regulated in tumors.

    PubMed

    Uekita, Takamasa; Gotoh, Isamu; Kinoshita, Takeshi; Itoh, Yoshifumi; Sato, Hiroshi; Shiomi, Takayuki; Okada, Yasunori; Seiki, Motoharu

    2004-03-26

    Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is an enzyme that promotes tumor cell invasion in tissues. Although the proteolytic activity of MT1-MMP is indispensable for invasion, it is also regulated by functions of the cytoplasmic tail. In this study we obtained a new human gene whose product binds to the tail sequence in yeast. The product, MTCBP-1, is a 19-kDa protein that belongs to the newly proposed Cupin superfamily composed of proteins with diverse functions. MTCBP-1 expressed in cells formed a complex with MT1-MMP and co-localized at the membrane. It was also detected in both the cytoplasm and nucleus, where MT1-MMP does not exist. In human tumor cell lines MTCBP-1 expression was significantly low compared with non-transformed fibroblasts, and enforced expression of MTCBP-1 inhibited the activity of MT1-MMP in promoting cell migration and invasion. MTCBP-1 showed significant homology to the bacterial aci-reductone dioxygenase, which is an enzyme in methionine metabolism. The C-terminal part of MTCBP-1 is identical to Sip-L, which is reported to be important for human hepatitis C virus replication. Thus, MTCBP-1 may have multiple functions other than the regulation of MT1-MMP, which presumably depends on the subcellular compartment.

  5. Characterization and Regulation of Suppressor of Cytokine Signaling (SOCS) Genes in Yellow Perch (Perca flavescens)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The suppressor of cytokine signaling (SOCS) proteins are a family of intracellular proteins that are centrally involved with vertebrate growth, development, and immunity via their effects as negative feedback regulators of cytokine (and hormone) signaling. A number of SOCS genes have been recently ...

  6. Myeloid Derived Suppressor Cells in Breast Cancer

    PubMed Central

    Markowitz, Joseph; Wesolowski, Robert; Papenfuss, Tracey; Brooks, Taylor R.

    2013-01-01

    Myeloid Derived Suppressor Cells (MDSCs) are a population of immature myeloid cells defined by their suppressive actions on immune cells such as T cells, dendritic cells, and natural killer cells. MDSCs typically are positive for the markers CD33 and CD11b but express low levels of HLADR in humans. In mice, MDSCs are typically positive for both CD11b and Gr1. These cells exert their suppressive activity on the immune system via the production of reactive oxygen species, arginase, and cytokines. These factors subsequently inhibit the activity of multiple protein targets such as the T cell receptor, STAT1, and indoleamine-pyrrole 2,3-dioxygenase. The numbers of MDSCs tend to increase with cancer burden while inhibiting MDSCs improves disease outcome in murine models. MDSCs also inhibit immune cancer therapeutics. In light of the poor prognosis of metastatic breast cancer in women and the correlation of increasing levels of MDSCs with increasing disease burden, the purposes of this review are to 1) discuss why MDSCs may be important in breast cancer, 2) describe model systems used to study MDSCs in vitro and in vivo, 3) discuss mechanisms involved in MDSC induction/function in breast cancer, and 4) present pre-clinical and clinical studies that explore modulation of the MDSC-immune system interaction in breast cancer. MDSCs inhibit the host immune response in breast cancer patients and diminishing MDSC actions may improve therapeutic outcomes. PMID:23828498

  7. Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

    PubMed

    Haverkamp, Jessica M; Smith, Amber M; Weinlich, Ricardo; Dillon, Christopher P; Qualls, Joseph E; Neale, Geoffrey; Koss, Brian; Kim, Young; Bronte, Vincenzo; Herold, Marco J; Green, Douglas R; Opferman, Joseph T; Murray, Peter J

    2014-12-18

    Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.

  8. Suppressor cells in transplantation tolerance. II. maturation of suppressor cells in the bone marrow chimera

    SciTech Connect

    Tutschka, P.J.; Ki, P.F.; Beschorner, W.E.; Hess, A.D.; Santos, G.W.

    1981-10-01

    Histoincompatible bone marrow allografts were established in lethally irradiated rats. At various times after transplantation, the spleen cells were harvested, subjected to mixed lymphocyte cultures, and assayed for suppressor cells in vitro and in vivo by adoptive transfer studies. Alloantigen-nonspecific suppressor cells appeared in the chimera at 40 days after grafting, coinciding with the resolution of graft-versus-host disease (GVHD). At 250 days the nonspecific suppressor cells were replaced by suppressor cells specifically suppressing donor-versus-host alloantigen responses. At 720 days suppressor cells could no longer be identified by in vitro methods but were identified by in vivo adoptive transfer of transplantation tolerance. After injection of host-type antigen into chimeras, the suppressor cells could be again demonstrated by in vitro methods.

  9. Suppressor cells in transplantation tolerance II. Maturation of suppressor cells in the bone marrow chimera

    SciTech Connect

    Tutschka, P.J.; Ki, P.F.; Beschorner, W.E.; Hess, A.D.; Santos, G.W.

    1981-10-01

    Histoincompatible bone marrow allografts were established in lethally irradiated rats. At various times after transplantation, the spleen cells were harvested, subjected to mixed lymphocyte cultures, and assayed for suppressor cells in vitro and in vivo by adoptive transfer studies. Alloantigen-nonspecific suppressor cells appeared in the chimera at 40 days after grafting, coinciding with the resolution of graft-versus-host disease (GVHD). At 250 days the nonspecific suppressor cells were replaced by suppressor cells specifically suppressing donor-versus-host alloantigen responses. At 720 days suppressor cells could no longer be identified by in vitro methods but were identified by in vivo adoptive transfer of transplantation tolerance. After injection of host-type antigen into chimeras, the suppressor cells could be again demonstrated by in vitro methods.

  10. Suppressors of cytokine signalling-3 and -1 in human carcinogenesis.

    PubMed

    Culig, Zoran

    2013-01-01

    The role of suppressors of cytokine signaling (SOCS)-3 and -1 has been investigated in various cancers. These proteins have been identified as endogenous controllers of activation of Janus kinase/signal transducers and activators of transcription factors pathway factors under physiological conditions and in disease. SOCS-3 expression is lost in several cancers due to epigenetic mechanisms, mostly promoter methylation. In liver, lung, and squamous head and neck cancer, and several hematological malignancies SOCS-3 acts as a classic tumor suppressor. In prostate cancer, SOCS-3 effects are cell type-dependent. It prevents apoptosis in androgen receptor-negative cells. However, in androgen-sensitive cells, it could act as a negative feedback factor for androgenic regulation. Melanoma cells which overexpress SOCS-3 confer a growth advantage. SOCS-1 is in most cancers a tumor suppressor which may inhibit expression of cyclin-dependent kinases and cyclins. In general, the mechanisms responsible for the different effects of SOCS in cancer cell lines have to be further investigated. The results discussed in the present review may have an impact on personalized approaches in cancer medicine. PMID:23277051

  11. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    PubMed

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. PMID:18533829

  12. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    PubMed

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

  13. Maspin as a Tumour Suppressor in Salivary Gland Tumour

    PubMed Central

    Ashok, Nipun; Sheirawan, Mohammad Kinan; Altamimi, Mohammed Alsakran; Alenzi, Faris; Azzeghaiby, Saleh Nasser; Baroudi, Kusai; Nassani, Mohammad Zakaria

    2014-01-01

    Maspin is a protein that belongs to serin protease inhibitor (serpin) superfamily. The purpose of this study was to review the literature concerning the expression of maspin in salivary gland tumours. A literature search was done using MEDLINE, accessed via the National Library of Medicine PubMed interface. Statistical analysis was not done because only seven studies were available in literature, the collected data were different and the results could not be compared. Expression of maspin was down regulated in more aggressive salivary gland tumours. Maspin may function as a tumour suppressor in salivary gland tumours. PMID:25654053

  14. The contrasting oncogenic and tumor suppressor roles of FES.

    PubMed

    Greer, Peter A; Kanda, Shigeru; Smithgall, Thomas E

    2012-01-01

    The FES gene was first discovered as a protein-tyrosine kinase-encoding retroviral oncogene. The ability of v-FES to transform cells in vitro and initiate cancer in vivo has been established by cell culture, engraftment and transgenic mouse studies. The corresponding cellular c-FES proto-oncogene encodes a cytoplasmic FES protein-tyrosine kinase with restrained catalytic activity relative to its retrovirally encoded homologs. These observations have stimulated a search for mutations or inappropriate expression of c-FES in human cancers and research aimed at understanding the functions of the FES kinase and its potential involvement in cancer and other diseases. Paradoxically, although first identified as an oncogene, genetic evidence has also implicated c-fes as a potential tumor suppressor. This review will describe observations from basic and translational research which shapes our current understanding of the physiological, cellular and molecular functions of the FES protein-tyrosine kinase and its potential roles in tumorigenesis. We also propose a model to reconcile the conflicting oncogenic and tumor suppressor roles of c-FES in tumorigenesis.

  15. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

    Kärblane, Kairi; Gerassimenko, Jelena; Nigul, Lenne; Piirsoo, Alla; Smialowska, Agata; Vinkel, Kadri; Kylsten, Per; Ekwall, Karl; Swoboda, Peter; Truve, Erkki; Sarmiento, Cecilia

    2015-01-01

    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. PMID:25659154

  16. [hScrib: a potential novel tumor suppressor].

    PubMed

    Borg, J-P

    2004-07-01

    Establishment and maintenance of epithelial cell polarity rely on finely tuned protein networks comprising cell surface molecules, cytoplasmic adaptors, and enzymes connected to the actin cytoskeleton. Oncogenes and tumor suppressors promote cell proliferation and resistance to apoptosis and, in many cases, alter some of these molecular scaffolds, and profoundly affect the epithelial cytoarchitecture. Reciprocally, loss of central actors of epithelial polarity unleashes normally repressed signaling pathways and perturb the shape and functions of epithelial tissues. Among the newcomers impacting on epithelial integrity, Scribble is a scaffold protein of a remarkable importance that furthermore displays a tumor suppressing activity in Drosophila melanogaster. Together with Discs Large (Dlg) and Lethal Giant Larvae (Lgl), two known tumor suppressors, Scribble acts on the correct positioning of epithelial junctions required to organize functional epithelial sheets. Scribble, Dlg and Lgl proteins are well conserved during evolution at the molecular and subcellular level implying their potential role in cell polarity and tumorigenesis in humans. Recent findings on hScrib, the human orthologue of Scribble, are discussed here.

  17. A novel plasmid-based microarray screen identifies suppressors of rrp6Delta in Saccharomyces cerevisiae.

    PubMed

    Abruzzi, Katharine; Denome, Sylvia; Olsen, Jens Raabjerg; Assenholt, Jannie; Haaning, Line Lindegaard; Jensen, Torben Heick; Rosbash, Michael

    2007-02-01

    Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Delta temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Delta strains at 37 degrees C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Delta strains. Microarray analyses of gene expression in rrp6Delta strains and a number of suppressor strains support this hypothesis. PMID:17101774

  18. Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

    PubMed

    Marinova, Irina N; Engelbrecht, Jacob; Ewald, Adrian; Langholm, Lasse L; Holmberg, Christian; Kragelund, Birthe B; Gordon, Colin; Nielsen, Olaf; Hartmann-Petersen, Rasmus

    2015-01-01

    The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors. PMID:25658828

  19. Single Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48 Identified by Whole Genome Sequencing

    PubMed Central

    Marinova, Irina N.; Engelbrecht, Jacob; Ewald, Adrian; Langholm, Lasse L.; Holmberg, Christian; Kragelund, Birthe B.; Gordon, Colin; Nielsen, Olaf; Hartmann-Petersen, Rasmus

    2015-01-01

    The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors. PMID:25658828

  20. Single site suppressors of a fission yeast temperature-sensitive mutant in cdc48 identified by whole genome sequencing.

    PubMed

    Marinova, Irina N; Engelbrecht, Jacob; Ewald, Adrian; Langholm, Lasse L; Holmberg, Christian; Kragelund, Birthe B; Gordon, Colin; Nielsen, Olaf; Hartmann-Petersen, Rasmus

    2015-01-01

    The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.

  1. Identification of ZDHHC14 as a novel human tumour suppressor gene.

    PubMed

    Yeste-Velasco, Marc; Mao, Xueying; Grose, Richard; Kudahetti, Sakunthala C; Lin, Dongmei; Marzec, Jacek; Vasiljević, Natasa; Chaplin, Tracy; Xue, Liyan; Xu, Maojia; Foster, Julie M; Karnam, Santi S; James, Sharon Y; Chioni, Athina-Myrto; Gould, David; Lorincz, Attila T; Oliver, R Tim D; Chelala, Claude; Thomas, Gareth M; Shipley, Janet M; Mather, Stephen J; Berney, Daniel M; Young, Bryan D; Lu, Yong-Jie

    2014-04-01

    Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer. PMID:24407904

  2. Transcriptional Regulation of the p16 Tumor Suppressor Gene.

    PubMed

    Kotake, Yojiro; Naemura, Madoka; Murasaki, Chihiro; Inoue, Yasutoshi; Okamoto, Haruna

    2015-08-01

    The p16 tumor suppressor gene encodes a specific inhibitor of cyclin-dependent kinase (CDK) 4 and 6 and is found altered in a wide range of human cancers. p16 plays a pivotal role in tumor suppressor networks through inducing cellular senescence that acts as a barrier to cellular transformation by oncogenic signals. p16 protein is relatively stable and its expression is primary regulated by transcriptional control. Polycomb group (PcG) proteins associate with the p16 locus in a long non-coding RNA, ANRIL-dependent manner, leading to repression of p16 transcription. YB1, a transcription factor, also represses the p16 transcription through direct association with its promoter region. Conversely, the transcription factors Ets1/2 and histone H3K4 methyltransferase MLL1 directly bind to the p16 locus and mediate p16 induction during replicative and premature senescence. In the present review, we discuss the molecular mechanisms by which these factors regulate p16 transcription.

  3. Non genomic loss of function of tumor suppressors in CML: BCR-ABL promotes IκBα mediated p53 nuclear exclusion.

    PubMed

    Crivellaro, Sabrina; Panuzzo, Cristina; Carrà, Giovanna; Volpengo, Alessandro; Crasto, Francesca; Gottardi, Enrico; Familiari, Ubaldo; Papotti, Mauro; Torti, Davide; Piazza, Rocco; Redaelli, Sara; Taulli, Riccardo; Guerrasio, Angelo; Saglio, Giuseppe; Morotti, Alessandro

    2015-09-22

    Tumor suppressor function can be modulated by subtle variation of expression levels, proper cellular compartmentalization and post-translational modifications, such as phosphorylation, acetylation and sumoylation. The non-genomic loss of function of tumor suppressors offers a challenging therapeutic opportunity. The reactivation of a tumor suppressor could indeed promote selective apoptosis of cancer cells without affecting normal cells. The identification of mechanisms that affect tumor suppressor functions is therefore essential. In this work, we show that BCR-ABL promotes the accumulation of the NFKBIA gene product, IκBα, in the cytosol through physical interaction and stabilization of the protein. Furthermore, BCR-ABL/IκBα complex acts as a scaffold protein favoring p53 nuclear exclusion. We therefore identify a novel BCR-ABL/IκBα/p53 network, whereby BCR-ABL functionally inactivates a key tumor suppressor. PMID:26295305

  4. Unique pathway of expression of an opal suppressor phosphoserine tRNA.

    PubMed Central

    Lee, B J; de la Peña, P; Tobian, J A; Zasloff, M; Hatfield, D

    1987-01-01

    An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. We have shown that the product of this gene functions as a suppressor in an in vitro assay, and we have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, we show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of any known eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells. Images PMID:3114749

  5. Unique pathway of expression of an opal suppressor phosphoserine tRNA

    SciTech Connect

    Lee, B.J.; De La Pena, P.; Tobian, J.A.; Zasloff, M.; Hatfield, D.

    1987-09-01

    An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. The authors have shown that the product of this gene functions as a suppressor in an in vitro assay, and they have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, they show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of nay know eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells.

  6. A natural polymorphism in β-lactamase is a global suppressor

    PubMed Central

    Huang, Wanzhi; Palzkill, Timothy

    1997-01-01

    A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 β-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in β-lactamase. The M182T substitution also is found in natural variants of TEM-1 β-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease. PMID:9238058

  7. Screening and identification of virus-encoded RNA silencing suppressors.

    PubMed

    Karjee, Sumona; Islam, Mohammad Nurul; Mukherjee, Sunil K

    2008-01-01

    RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities.

  8. Studies Identify Non-Hodgkin Lymphoma Suppressor.

    PubMed

    2015-12-01

    Two new studies show that the histone methyltransferase KMT2D, known to be frequently mutated in the two most common forms of non-Hodgkin lymphoma, is a bona fide tumor suppressor. KMT2D mutations are loss-of-function events that remodel the epigenetic landscape of developing B cells, predisposing them toward malignancy. PMID:26463831

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

    PubMed Central

    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

  10. Muscleblind-like 1 suppresses breast cancer metastatic colonization and stabilizes metastasis suppressor transcripts

    PubMed Central

    Fish, Lisa; Pencheva, Nora; Goodarzi, Hani; Tran, Hien; Yoshida, Mitsukuni; Tavazoie, Sohail F.

    2016-01-01

    Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3′ untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)—two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes’ transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization. PMID:26883358

  11. Muscleblind-like 1 suppresses breast cancer metastatic colonization and stabilizes metastasis suppressor transcripts.

    PubMed

    Fish, Lisa; Pencheva, Nora; Goodarzi, Hani; Tran, Hien; Yoshida, Mitsukuni; Tavazoie, Sohail F

    2016-02-15

    Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3' untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)-two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes' transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization.

  12. Jade-1, a candidate renal tumor suppressor that promotes apoptosis.

    PubMed

    Zhou, Mina I; Foy, Rebecca L; Chitalia, Vipul C; Zhao, Jin; Panchenko, Maria V; Wang, Hongmei; Cohen, Herbert T

    2005-08-01

    Medical therapies are lacking for advanced renal cancer, so there is a great need to understand its pathogenesis. Most renal cancers have defects in the von Hippel-Lindau tumor suppressor pVHL. The mechanism by which pVHL protein functions in renal tumor suppression remains unclear. Jade-1 is a short-lived, kidney-enriched transcription factor that is stabilized by direct interaction with pVHL. Loss of Jade-1 stabilization by pVHL correlates with renal cancer risk, making the relationship between Jade-1 and renal cancer compelling. We report that Jade-1 expression was barely detectable in all tested renal cancer cell lines, regardless of VHL status. Strikingly, proteasome inhibitor treatment increased endogenous Jade-1 expression up to 10-fold. Jade-1 inhibited renal cancer cell growth, colony formation, and tumor formation in nude mice. Intriguingly, Jade-1 also affected the pattern of cell growth in monolayer culture and 3D culture. Jade-1 increased apoptosis by 40-50% and decreased levels of antiapoptotic Bcl-2. Antisense Jade-1-expressing cells confirmed these results. Therefore, Jade-1 may suppress renal cancer cell growth in part by increasing apoptosis. Jade-1 may represent a proapoptotic barrier to proliferation that must be overcome generally in renal cancer, perhaps initially by pVHL inactivation and subsequently by increased proteasomal activity. Therefore, Jade-1 may be a renal tumor suppressor.

  13. Subpopulation of human helper and suppressor T lymphocytes

    SciTech Connect

    Venkataraman, M.; Levin, R.D.; Westerman, M.P.

    1983-07-01

    Mitogen driven differentiation of normal human mononuclear cells is a well-established model for the study of antibody synthesis in man. In certain rare individuals who are clinically normal, unfractionated mononuclear cells or a mixture of purified B plus T lymphocytes differentiate into immunoglobulin producing cells in response to purified protein derivative of tuberculin (PPD) but not in response to pokeweed mitogen (PWM). To evaluate this observation we have irradiated T cells from such individuals to eliminate naturally occurring suppressor T cell activity and then added the irradiated T cells back to autologous B cells before culture. The B cells then responded to PWM. The original PPD responses of cells from these individuals were now significantly reduced. Although, there was no difference between PWM nonresponders and responders in the number of OKT-8 positive cells, elimination of OKT-8 positive cells in the PWM nonresponders with OKT-8 monoclonal antibody and complement resulted in a significantly increased response to PWM. This study indicates that there are suppressor T cells which specifically inhibit B cell response to PWM without affecting the PPD response. These results also show that the helper T cells involved in the PWM response are radioresistant and those involved in the PPD response are radiosensitive.

  14. An unusual characteristic "flower-like" pattern: flash suppressor burns.

    PubMed

    Gurcan, Altun

    2012-04-01

    The case on contact shots from firearms with a flash suppressor is rare. When a rifle fitted with a flash suppressor is fired, the emerging soot-laden gas in the barrel escapes from the slits of the flash suppressor. If the shot is contact or near contact, the flash suppressor will produce a characteristic "flower-like" pattern of seared, blackened zones around the entrance. This paper presents the injury pattern of the flash suppressor in a 29-year-old man who committed suicide with a G3 automatic infantry rifle.

  15. Isolation and Characterization of Suppressors of Two Escherichia Coli Dnag Mutations, Dnag2903 and Parb

    PubMed Central

    Britton, R. A.; Lupski, J. R.

    1997-01-01

    The dnaG gene of Escherichia coli encodes the primase protein, which synthesizes a short pRNA that is essential for the initiation of both leading and lagging strand DNA synthesis. Two temperature-sensitive mutations in the 3' end of the dnaG gene, dnaG2903 and parB, cause a defect in chromosome partitioning at the nonpermissive temperature 42°. We have characterized 24 cold-sensitive suppressor mutations of these two dnaG alleles. By genetic mapping and complementation, five different classes of suppressors have been assigned: sdgC, sdgD, sdgE, sdgG and sdgH. The genes responsible for suppression in four of the five classes have been determined. Four of the sdgC suppressor alleles are complemented by the dnaE gene, which encodes the enzymatic subunit of DNA polymerase III. The sdgE class are mutations in era, an essential GTPase of unknown function. The sdgG suppressor is likely a mutation in one of three genes: ubiC, ubiA or yjbI. The sdgH class affects rpsF, which encodes the ribosomal protein S6. Possible mechanisms of suppression by these different classes are discussed. PMID:9093842

  16. Power of PTEN/AKT: Molecular switch between tumor suppressors and oncogenes

    PubMed Central

    XIE, YINGQIU; NAIZABEKOV, SANZHAR; CHEN, ZHANLIN; TOKAY, TURSONJAN

    2016-01-01

    An increasing amount of evidence has shown that tumor suppressors can become oncogenes, or vice versa, but the mechanism behind this is unclear. Recent findings have suggested that phosphatase and tensin homolog (PTEN) is one of the powerful switches for the conversion between tumor suppressors and oncogenes. PTEN regulates a number of cellular processes, including cell death and proliferation, through the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Furthermore, a number of studies have suggested that PTEN deletions may alter various functions of certain tumor suppressor and oncogenic proteins. The aim of the present review was to analyze specific cases driven by PTEN loss/AKT activation, including aberrant signaling pathways and novel drug targets for clinical application in personalized medicine. The findings illustrate how PTEN loss and/or AKT activation switches MDM2-dependent p53 downregulation, and induces conversion between oncogene and tumor suppressor in enhancer of zeste homolog 2, BTB domain-containing 7A, alternative reading frame 2, p27 and breast cancer 1, early onset, through multiple mechanisms. This review highlights the genetic basis of complex drug targets and provides insights into the rationale of precision cancer therapy. PMID:27347153

  17. BRIT1 regulates p53 stability and functions as a tumor suppressor in breast cancer

    PubMed Central

    Zhang, Bo; Wang, Edward; Lin, Shiaw-Yih

    2013-01-01

    In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1’s tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer. PMID:23729656

  18. LKB1, the multitasking tumour suppressor kinase.

    PubMed

    Marignani, P A

    2005-01-01

    Mutations in the lkb1 gene are found in Peutz-Jeghers syndrome (PJS), with loss of heterozygosity or somatic mutations at the lkb1 locus, suggesting the gene product, the serine/threonine kinase LKB1, may function as a tumour suppressor. Patients with PJS are at a greater risk of developing cancers of epithelial tissue origin. It is widely accepted that the presence of hamartomatous polyps in PJS does not in itself lead to the development of malignancy. The signalling mechanisms that lead to these PJS related malignancies are not well understood. However, it is evident from the recent literature that LKB1 is a multitasking kinase, with unlimited potential in orchestrating cell activity. Thus far, LKB1 has been found to play a role in chromatin remodelling, cell cycle arrest, Wnt signalling, cell polarity, and energy metabolism, all of which may require the tumour suppressor function of this kinase and/or its catalytic activity.

  19. PAQR3: a novel tumor suppressor gene

    PubMed Central

    Yu, Xin; Li, Zheng; Chan, Matthew TV; Wu, William Ka Kei

    2015-01-01

    PAQR3, also known as RKTG (Raf kinase trapping to Golgi), is a member of the progestin and adipoQ receptor (PAQR) family. The role of PAQR3 as a tumor suppressor has recently been established in different types of human cancer in which PAQR3 exerts its biological function through negative regulation of the oncogenic Raf/MEK/ERK signaling. Multiple studies have found that PAQR3 downregulation frequently occurs in human cancers and is very often associated with tumor progression and shortened patients’ survival. Moreover, restoring the expression of PAQR3 could induce apoptosis and inhibit proliferation and invasiveness of cancer cells. Downregulation of PAQR3 by oncogenic microRNAs has also been reported. In this review, we summarized current knowledge concerning the role of PAQR3 in tumor development. To our knowledge, this is the first review on the role of this novel tumor suppressor. PMID:26609468

  20. Multielement suppressor nozzles for thrust augmentation systems.

    NASA Technical Reports Server (NTRS)

    Lawrence, R. L.; O'Keefe, J. V.; Tate, R. B.

    1972-01-01

    The noise reduction and nozzle performance characteristics of large-scale, high-aspect-ratio multielement nozzle arrays operated at low velocities were determined by test. The nozzles are selected for application to high-aspect-ratio augmentor suppressors to be used for augmentor wing airplanes. Significant improvements in noise characteristics for multielement nozzles over those of round or high-aspect-ratio slot nozzles are obtained. Elliptical noise patterns typical of slot nozzles are presented for high-aspect-ratio multielement nozzle arrays. Additional advantages are available in OASPL noise reduction from the element size and spacing. Augmentor-suppressor systems can be designed for maximum beam pattern directivity and frequency spectrum shaping advantages. Measurements of the nozzle wakes show a correlation with noise level data and frequency spectrum peaks. The noise and jet wake results are compared with existing prediction procedures based on empirical jet flow equations, Lighthill relationships, Strouhal number, and empirical shock-induced screech noise effects.

  1. Caspase-2 as a tumour suppressor

    PubMed Central

    Puccini, J; Dorstyn, L; Kumar, S

    2013-01-01

    Ever since its discovery 20 years ago, caspase-2 has been enigmatic and its function somewhat controversial. Although many in vitro studies suggested that caspase-2 was important for apoptosis, demonstrating an in vivo cell death role for this caspase has been more problematic, with caspase-2-deficient mice showing limited, tissue-specific cell death defects. Recent results from different laboratories suggest that at least one of its physiological roles in animals is to protect against cellular stress and transformation. As such, loss of caspase-2 augments tumorigenesis in some mouse models of cancer, assigning a tumour suppressor function to this enigmatic caspase. This review focuses on this seemingly non-apoptotic function of caspase-2 as a tumour suppressor and reconciles some of the recent findings in the field. PMID:23811850

  2. Tet1 is a tumor suppressor of hematopoietic malignancy

    PubMed Central

    Cimmino, Luisa; Dawlaty, Meelad M.; Ndiaye-Lobry, Delphine; Yap, Yoon Sing; Bakogianni, Sofia; Yu, Yiting; Bhattacharyya, Sanchari; Shaknovich, Rita; Geng, Huimin; Lobry, Camille; Mullenders, Jasper; King, Bryan; Trimarchi, Thomas; Aranda-Orgilles, Beatriz; Liu, Cynthia; Shen, Steven; Verma, Amit K.; Jaenisch, Rudolf; Aifantis, Iannis

    2015-01-01

    The TET methylcytosine dioxygenase 1 (TET1) enzyme is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. Decreased expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we show that deletion of Tet1 promoted the development of B cell lymphoma in mice. Tet1 was required for maintaining normal content of 5hmC, preventing DNA hypermethylation and in the regulation of B cell lineage, chromosome maintenance and DNA repair genes. Whole-exome sequencing of Tet1-deficient tumors revealed mutations frequently found in Non-Hodgkin B cell lymphoma, where TET1 was hypermethylated and transcriptionally silenced. These findings provide in vivo evidence of TET1 function as a tumor suppressor of hematopoietic malignancy. PMID:25867473

  3. Identification of an RNA silencing suppressor encoded by a mastrevirus.

    PubMed

    Wang, Yaqin; Dang, Mingqing; Hou, Huwei; Mei, Yuzhen; Qian, Yajuan; Zhou, Xueping

    2014-09-01

    Wheat dwarf virus (WDV) is a DNA virus belonging to the genus Mastrevirus of the family Geminiviridae. In this study, we report that the Rep protein encoded by WDV is a RNA silencing supressor as determined by co-infiltration assays using transgenic Nicotiana benthamiana line 16c carrying the GFP reporter gene. The Rep protein was shown to inhibit both local and systemic RNA silencing of the GFP gene as well as the spread of systemic GFP RNA silencing signals. Gel mobility shift assays showed that the Rep protein binds 21 nt and 24 nt small interfering RNA (siRNA) duplexes and single-stranded (ss)-siRNA. To our knowledge, this is the first identification of an RNA silencing suppressor encoded by mastreviruses. Furthermore, deletion mutagenesis indicates that both the N- and C-terminal regions of the Rep protein are not critical for silencing suppression and self-interaction, but the N terminus of Rep is necessary for its pathogenicity. PMID:24866851

  4. Insulation of Enhancer-Promoter Communication by a Gypsy Transposon Insert in the Drosophila cut Gene: Cooperation between Suppressor of Hairy-wing and Modifier of mdg4 Proteins

    PubMed Central

    Gause, Maria; Morcillo, Patrick; Dorsett, Dale

    2001-01-01

    The Drosophila mod(mdg4) gene products counteract heterochromatin-mediated silencing of the white gene and help activate genes of the bithorax complex. They also regulate the insulator activity of the gypsy transposon when gypsy inserts between an enhancer and promoter. The Su(Hw) protein is required for gypsy-mediated insulation, and the Mod(mdg4)-67.2 protein binds to Su(Hw). The aim of this study was to determine whether Mod(mdg4)-67.2 is a coinsulator that helps Su(Hw) block enhancers or a facilitator of activation that is inhibited by Su(Hw). Here we provide evidence that Mod(mdg4)-67.2 acts as a coinsulator by showing that some loss-of-function mod(mdg4) mutations decrease enhancer blocking by a gypsy insert in the cut gene. We find that the C terminus of Mod(mdg4)-67.2 binds in vitro to a region of Su(Hw) that is required for insulation, while the N terminus mediates self-association. The N terminus of Mod(mdg4)-67.2 also interacts with the Chip protein, which facilitates activation of cut. Mod(mdg4)-67.2 truncated in the C terminus interferes in a dominant-negative fashion with insulation in cut but does not significantly affect heterochromatin-mediated silencing of white. We infer that multiple contacts between Su(Hw) and a Mod(mdg4)-67.2 multimer are required for insulation. We theorize that Mod(mdg4)-67.2 usually aids gene activation but can also act as a coinsulator by helping Su(Hw) trap facilitators of activation, such as the Chip protein. PMID:11416154

  5. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    PubMed Central

    Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine. PMID:27529623

  6. Tuning of alternative splicing--switch from proto-oncogene to tumor suppressor.

    PubMed

    Shchelkunova, Aleksandra; Ermolinsky, Boris; Boyle, Meghan; Mendez, Ivan; Lehker, Michael; Martirosyan, Karen S; Kazansky, Alexander V

    2013-01-01

    STAT5B, a specific member of the STAT family, is intimately associated with prostate tumor progression. While the full form of STAT5B is thought to promote tumor progression, a naturally occurring truncated isoform acts as a tumor suppressor. We previously demonstrated that truncated STAT5 is generated by insertion of an alternatively spliced exon and results in the introduction of an early termination codon. Present approaches targeting STAT proteins based on inhibition of functional domains of STAT's, such as DNA-binding, cooperative binding (protein-protein interaction), dimerization and phosphorylation will halt the action of the entire gene, both the proto-oncogenic and tumor suppressor functions of Stat5B. In this report we develop a new approach aimed at inhibiting the expression of full-length STAT5B (a proto-oncogene) while simultaneously enhancing the expression of STAT5∆B (a tumor suppressor). We have demonstrated the feasibility of using steric-blocking splice-switching oligonucleotides (SSOs) with a complimentary sequence to the targeted exon-intron boundary to enhance alternative intron/exon retention (up to 10%). The functional effect of the intron/exon proportional tuning was validated by cell proliferation and clonogenic assays. The new scheme applies specific steric-blocking splice-switching oligonucleotides and opens an opportunity for anti-tumor treatment as well as for the alteration of functional abilities of other STAT proteins.

  7. The Tumor Suppressor rpl36 Restrains KRASG12V-Induced Pancreatic Cancer

    PubMed Central

    Provost, Elayne; Bailey, Jennifer M.; Aldrugh, Sumar; Liu, Shu; Iacobuzio-Donahue, Christine

    2014-01-01

    Abstract Ribosomal proteins are known to be required for proper assembly of mature ribosomes. Recent studies indicate an additional role for ribosomal proteins as candidate tumor suppressor genes. Pancreatic acinar cells, recently identified as effective cells of origin for pancreatic adenocarcinoma, display especially high-level expression of multiple ribosomal proteins. We, therefore, functionally interrogated the ability of two ribosomal proteins, rpl36 and rpl23a, to alter the response to oncogenic Kras in pancreatic acinar cells using a newly established model of zebrafish pancreatic cancer. These studies reveal that rpl36, but not rpl23a, acts as a haploinsufficient tumor suppressor, as manifested by more rapid tumor progression and decreased survival in rpl36hi1807/+;ptf1a:gal4VP16Tg;UAS:GFP-KRASG12V fish compared with their rpl36+/+;ptf1a:gal4VP16;UAS:GFP-KRASG12V siblings. These results suggest that rpl36 may function as an effective tumor suppressor during pancreatic tumorigenesis. PMID:25380065

  8. Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

    PubMed Central

    Kontra, Levente; Tavazza, Mario; Lucioli, Alessandra; Tavazza, Raffaela; Moxon, Simon; Medzihradszky, Anna; Burgyán, József

    2016-01-01

    RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. PMID:27711201

  9. Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

    PubMed Central

    Rodriguez, Erik A.; Lester, Henry A.; Dougherty, Dennis A.

    2007-01-01

    The incorporation of unnatural amino acids site-specifically is a valuable technique for structure–function studies, incorporation of biophysical probes, and determining protein–protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells. PMID:17698638

  10. A Novel Plasmid-Based Microarray Screen Identifies Suppressors of rrp6Δ in Saccharomyces cerevisiae▿†

    PubMed Central

    Abruzzi, Katharine; Denome, Sylvia; Olsen, Jens Raabjerg; Assenholt, Jannie; Haaning, Line Lindegaard; Jensen, Torben Heick; Rosbash, Michael

    2007-01-01

    Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Δ strains. Microarray analyses of gene expression in rrp6Δ strains and a number of suppressor strains support this hypothesis. PMID:17101774

  11. Tumour suppressor TRIM33 targets nuclear β-catenin degradation

    PubMed Central

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

    2014-01-01

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

  12. Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway

    PubMed Central

    Fang, Su-Chiung; de los Reyes, Chris; Umen, James G

    2006-01-01

    Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription. PMID:17040130

  13. Tumor suppressor properties of the splicing regulatory factor RBM10

    PubMed Central

    Hernández, Jordi; Bechara, Elias; Schlesinger, Doerte; Delgado, Javier; Serrano, Luis; Valcárcel, Juan

    2016-01-01

    ABSTRACT RBM10 is an RNA binding protein and alternative splicing regulator frequently mutated in lung adenocarcinomas. Recent results indicate that RBM10 inhibits proliferation of lung cancer cells by promoting skipping of exon 9 of the gene NUMB, a frequent alternative splicing change in lung cancer generating a negative regulator of Notch signaling. Complementing these observations, we show that knock down of RBM10 in human cancer cells enhances growth of mouse tumor xenografts, confirming that RBM10 acts as a tumor suppressor, while knock down of an oncogenic mutant version of RBM10 reduces xenograft tumor growth. A RBM10 mutation found in lung cancer cells, V354E, disrupts RBM10-mediated regulation of NUMB alternative splicing, inducing the cell proliferation-promoting isoform. We now show that 2 natural RBM10 isoforms that differ by the presence or absence of V354 in the second RNA Recognition Motif (RRM2), display similar regulatory effects on NUMB alternative splicing, suggesting that V354E actively disrupts RBM10 activity. Structural modeling localizes V354 in the outside surface of one α-helix opposite to the RNA binding surface of RBM10, and we show that the mutation does not compromise binding of the RRM2 domain to NUMB RNA regulatory sequences. We further show that other RBM10 mutations found in lung adenocarcinomas also compromise regulation of NUMB exon 9. Collectively, our previous and current results reveal that RBM10 is a tumor suppressor that represses Notch signaling and cell proliferation through the regulation of NUMB alternative splicing. PMID:26853560

  14. Wilms Tumor Suppressor, WT1, Cooperates with MicroRNA-26a and MicroRNA-101 to Suppress Translation of the Polycomb Protein, EZH2, in Mesenchymal Stem Cells.

    PubMed

    Akpa, Murielle M; Iglesias, Diana; Chu, LeeLee; Thiébaut, Antonin; Jentoft, Ida; Hammond, Leah; Torban, Elena; Goodyer, Paul R

    2016-02-19

    Hereditary forms of Wilms arise from developmentally arrested clones of renal progenitor cells with biallelic mutations of WT1; recently, it has been found that Wilms tumors may also be associated with biallelic mutations in DICER1 or DROSHA, crucial for miRNA biogenesis. We have previously shown that a critical role for WT1 during normal nephrogenesis is to suppress transcription of the Polycomb group protein, EZH2, thereby de-repressing genes in the differentiation cascade. Here we show that WT1 also suppresses translation of EZH2. All major WT1 isoforms induce an array of miRNAs, which target the 3' UTR of EZH2 and other Polycomb-associated transcripts. We show that the WT1(+KTS) isoform binds to the 5' UTR of EZH2 and interacts directly with the miRNA-containing RISC to enhance post-transcriptional inhibition. These observations suggest a novel mechanism through which WT1 regulates the transition from resting stem cell to activated progenitor cell during nephrogenesis. Our findings also offer a plausible explanation for the fact that Wilms tumors can arise either from loss of WT1 or loss of miRNA processing enzymes. PMID:26655220

  15. A suppressor locus for MODY3-diabetes

    PubMed Central

    Garcia-Gonzalez, Miguel A.; Carette, Claire; Bagattin, Alessia; Chiral, Magali; Makinistoglu, Munevver Parla; Garbay, Serge; Prévost, Géraldine; Madaras, Cécile; Hérault, Yann; Leibovici, Michel; Pontoglio, Marco

    2016-01-01

    Maturity Onset Diabetes of the Young type 3 (MODY3), linked to mutations in the transcription factor HNF1A, is the most prevalent form of monogenic diabetes mellitus. HNF1alpha-deficiency leads to defective insulin secretion via a molecular mechanism that is still not completely understood. Moreover, in MODY3 patients the severity of insulin secretion can be extremely variable even in the same kindred, indicating that modifier genes may control the onset of the disease. With the use of a mouse model for HNF1alpha-deficiency, we show here that specific genetic backgrounds (C3H and CBA) carry a powerful genetic suppressor of diabetes. A genome scan analysis led to the identification of a major suppressor locus on chromosome 3 (Moda1). Moda1 locus contains 11 genes with non-synonymous SNPs that significantly interacts with other loci on chromosomes 4, 11 and 18. Mechanistically, the absence of HNF1alpha in diabetic-prone (sensitive) strains leads to postnatal defective islets growth that is remarkably restored in resistant strains. Our findings are relevant to human genetics since Moda1 is syntenic with a human locus identified by genome wide association studies of fasting glycemia in patients. Most importantly, our results show that a single genetic locus can completely suppress diabetes in Hnf1a-deficiency. PMID:27667715

  16. Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

    PubMed Central

    Nelson, M. K.; Kurihara, T.; Silver, P. A.

    1993-01-01

    Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function. PMID:8514125

  17. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

    NASA Astrophysics Data System (ADS)

    Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

    2004-11-01

    Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

  18. Compositional features are potentially involved in the regulation of gene expression of tumor suppressor genes in human tissues.

    PubMed

    Hajjari, Mohammadreza; Khoshnevisan, Atefeh; Behmanesh, Mehrdad

    2014-12-15

    Different mechanisms regulate the expression level of tissue specific genes in human. Here we report some compositional features such as codon usage bias, amino acid usage bias, codon frequency, and base composition which may be potentially related to mRNA amount of tissue specific tumor suppressor genes. Our findings support the possibility that structural elements in gene and protein may play an important role in the regulation of tumor suppressor genes, development, and tumorigenesis. The data presented here can open broad vistas in the understanding and treatment of a variety of human malignancies.

  19. Defining and Interpreting Suppressor Effects: Advantages and Limitations.

    ERIC Educational Resources Information Center

    Lancaster, Brian P.

    Suppressor effects are considered one of the most elusive dynamics in the interpretation of statistical data. A suppressor variable has been defined as a predictor that has a zero correlation with the dependent variable while still, paradoxically, contributing to the predictive validity of the test battery (P. Horst, 1941). This paper explores the…

  20. Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

    PubMed Central

    Köhrer, Caroline; Sullivan, Eric L.; RajBhandary, Uttam L.

    2004-01-01

    We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells. PMID:15576346

  1. Regulation of cell signaling and apoptosis by tumor suppressor WWOX

    PubMed Central

    Lo, Jui-Yen; Chou, Ying-Tsen; Lai, Feng-Jie

    2015-01-01

    Human fragile WWOX gene encodes a tumor suppressor WW domain-containing oxidoreductase (named WWOX, FOR, or WOX1). Functional suppression of WWOX prevents apoptotic cell death induced by a variety of stress stimuli, such as tumor necrosis factor, UV radiation, and chemotherapeutic drug treatment. Loss of WWOX gene expression due to gene deletions, loss of heterozygosity, chromosomal translocations, or epigenetic silencing is frequently observed in human malignant cancer cells. Acquisition of chemoresistance in squamous cell carcinoma, osteosarcoma, and breast cancer cells is associated with WWOX deficiency. WWOX protein physically interacts with many signaling molecules and exerts its regulatory effects on gene transcription and protein stability and subcellular localization to control cell survival, proliferation, differentiation, autophagy, and metabolism. In this review, we provide an overview of the recent advances in understanding the molecular mechanisms by which WWOX regulates cellular functions and stress responses. A potential scenario is that activation of WWOX by anticancer drugs is needed to overcome chemoresistance and trigger cancer cell death, suggesting that WWOX can be regarded as a prognostic marker and a candidate molecule for targeted cancer therapies. PMID:25595191

  2. Isolation of Omnipotent Suppressors in an [Eta(+)] Yeast Strain

    PubMed Central

    All-Robyn, J. A.; Kelley-Geraghty, D.; Griffin, E.; Brown, N.; Liebman, S. W.

    1990-01-01

    Omnipotent suppressors decrease translational fidelity and cause misreading of nonsense codons. In the presence of the non-Mendelian factor [eta(+)], some alleles of previously isolated omnipotent suppressors are lethal. Thus the current search was conducted in an [eta(+)] strain in an effort to identify new suppressor loci. A new omnipotent suppressor, SUP39, and alleles of sup35, sup45, SUP44 and SUP46 were identified. Efficiencies of the dominant suppressors were dramatically reduced in strains that were cured of non-Mendelian factors by growth on guanidine hydrochloride. Wild-type alleles of SUP44 and SUP46 were cloned and these clones were used to facilitate the genetic analyses. SUP44 was shown to be on chromosome VII linked to cyh2, and SUP46 was clearly identified as distinct from the linked sup45. PMID:2311916

  3. Dengue virus-specific suppressor T cells: current perspectives.

    PubMed

    Chaturvedi, Umesh C; Shrivastava, Richa; Tripathi, Raj K; Nagar, Rachna

    2007-08-01

    Dengue virus was the first microorganism that was shown to induce generation of antigen-specific suppressor T (TS) cells in mice. The cascade of the three generations of TS cells (TS1, TS2, TS3) and their secretary products, the suppressor factors (SF1, SF2), was delineated. The TS pathway was proposed to be protective through inhibition of the production of enhancing antibody, which may enhance the severity of dengue disease. The currently second most favoured mechanism of severe dengue disease is the 'cytokine tsunami'. During the last decade, suppressor/regulatory T cells have been studied in greater detail using modern techniques in various diseases, including viral infections. This brief review discusses the role of dengue-specific suppressor T cells in protection and/or induction of severe dengue disease in view of our current understanding of suppressor/regulatory T cells. PMID:17573929

  4. Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways

    PubMed Central

    Haverkamp, Jessica M.; Smith, Amber M.; Weinlich, Ricardo; Dillon, Christopher P.; Qualls, Joseph E.; Neale, Geoffrey; Koss, Brian; Kim, Young; Bronte, Vincenzo; Herold, Marco J.; Green, Douglas R.; Opferman, Joseph T.; Murray, Peter J.

    2014-01-01

    Summary Non-resolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for anti-tumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor cell subset, but not the granulocytic subset requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, while their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival and function of myeloid suppressor cells. PMID:25500368

  5. miR-152 as a tumor suppressor microRNA: Target recognition and regulation in cancer

    PubMed Central

    LIU, XUEXIANG; LI, JINWAN; QIN, FENGXIAN; DAI, SHENGMING

    2016-01-01

    MicroRNAs (miRNAs or miRs) are endogenous translation repressors of protein-coding genes that act by binding to the 3′-untranslated region of their target genes, and may contribute to tumorigenesis by functioning as oncogenes or tumor suppressor genes. miR-152, a member of the miR-148/152 family, is aberrantly expressed in various diseases, including various types of cancer. A growing body of evidence has demonstrated that miR-152 may act as a tumor suppressor gene by regulating its target genes, which are associated with cell proliferation, migration and invasion in human cancer. In the present review, the gene structure and functions of miR-152 are discussed, and in particular, its regulatory mechanism, experimentally validated targets and tumor suppressor role in cancer, are highlighted. PMID:27313716

  6. DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma

    PubMed Central

    Xue, Wen; Krasnitz, Alexander; Lucito, Robert; Sordella, Raffaella; VanAelst, Linda; Cordon-Cardo, Carlos; Singer, Stephan; Kuehnel, Florian; Wigler, Michael; Powers, Scott; Zender, Lars; Lowe, Scott W.

    2008-01-01

    Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically. PMID:18519636

  7. Myeloid derived suppressor cells and autoimmunity.

    PubMed

    Boros, Peter; Ochando, Jordi; Zeher, Margit

    2016-08-01

    Myeloid-derived suppressor cells are a heterogeneous group of immature myeloid cells with immunoregulatory function. When activated and expanded, these cells can suppress T cell functions via cell-to cell interactions as well as soluble mediators. Recent studies investigated the involvement of MDSC in autoimmune diseases. Some papers have described beneficial effect of MDSC during the course of autoimmune diseases, and suggest a potential role as a treatment option, while others failed to detect these effects. Their contributions to autoimmune diseases are not fully understood, and many questions and some controversies remain as to the expansion, activation, and inhibitory functions of MDSC. This review aims to summarize current knowledge of MDSC in autoimmune disorders. PMID:27240453

  8. A Forward Genetic Screen for Suppressors of Somatic P Granules in Caenorhabditis elegans

    PubMed Central

    Kelly, Ashley L.; Senter-Zapata, Michael J.; Campbell, Anne C.; Lust, Hannah E.; Theriault, Monique E.; Andralojc, Karolina M.; Updike, Dustin L.

    2015-01-01

    In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation. PMID:26100681

  9. LRIG1 is a triple threat: ERBB negative regulator, intestinal stem cell marker and tumour suppressor.

    PubMed

    Wang, Y; Poulin, E J; Coffey, R J

    2013-05-14

    In baseball parlance, a triple threat is a person who can run, hit and throw with aplomb. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a cell surface protein that antagonises ERBB receptor signalling by downregulating receptor levels. Over 10 years ago, Hedman et al postulated that LRIG1 might be a tumour suppressor. Recently, Powell et al provided in vivo evidence substantiating that claim by demonstrating that Lrig1 loss in mice leads to spontaneously arising, highly penetrant intestinal adenomas. Interestingly, Lrig1 also marks stem cells in the gut, suggesting a potential role for Lrig1 in maintaining intestinal epithelial homeostasis. In this review, we will discuss the ability of LRIG1 to act as a triple threat: pan-ERBB negative regulator, intestinal stem cell marker and tumour suppressor. We will summarise studies of LRIG1 expression in human cancers and discuss possible related roles for LRIG2 and LRIG3.

  10. Cancer-associated p53 tetramerization domain mutants: quantitative analysis reveals a low threshold for tumor suppressor inactivation

    SciTech Connect

    Kamada, R.; Anderson, C.; Nomura, T.; Sakaguchi, K.

    2011-01-07

    The tumor suppressor p53, a 393-amino acid transcription factor, induces cell cycle arrest and apoptosis in response to genotoxic stress. Its inactivation via the mutation of its gene is a key step in tumor progression, and tetramer formation is critical for p53 post-translational modification and its ability to activate or repress the transcription of target genes vital in inhibiting tumor growth. About 50% of human tumors have TP53 gene mutations; most are missense ones that presumably lower the tumor suppressor activity of p53. In this study, we explored the effects of known tumor-derived missense mutations on the stability and oligomeric structure of p53; our comprehensive, quantitative analyses encompassed the tetramerization domain peptides representing 49 such substitutions in humans. Their effects on tetrameric structure were broad, and the stability of the mutant peptides varied widely ({Delta}T{sub m} = 4.8 {approx} -46.8 C). Because formation of a tetrameric structure is critical for protein-protein interactions, DNA binding, and the post-translational modification of p53, a small destabilization of the tetrameric structure could result in dysfunction of tumor suppressor activity. We suggest that the threshold for loss of tumor suppressor activity in terms of the disruption of the tetrameric structure of p53 could be extremely low. However, other properties of the tetramerization domain, such as electrostatic surface potential and its ability to bind partner proteins, also may be important.

  11. ER functions of oncogenes and tumor suppressors: Modulators of intracellular Ca(2+) signaling.

    PubMed

    Bittremieux, Mart; Parys, Jan B; Pinton, Paolo; Bultynck, Geert

    2016-06-01

    Intracellular Ca(2+) signals that arise from the endoplasmic reticulum (ER), the major intracellular Ca(2+)-storage organelle, impact several mitochondrial functions and dictate cell survival and cell death processes. Furthermore, alterations in Ca(2+) signaling in cancer cells promote survival and establish a high tolerance towards cell stress and damage, so that the on-going oncogenic stress does not result in the activation of cell death. Over the last years, the mechanisms underlying these oncogenic alterations in Ca(2+) signaling have started to emerge. An important aspect of this is the identification of several major oncogenes, including Bcl-2, Bcl-XL, Mcl-1, PKB/Akt, and Ras, and tumor suppressors, such as p53, PTEN, PML, BRCA1, and Beclin 1, as direct and critical regulators of Ca(2+)-transport systems located at the ER membranes, including IP3 receptors and SERCA Ca(2+) pumps. In this way, these proteins execute part of their function by controlling the ER-mitochondrial Ca(2+) fluxes, favoring either survival (oncogenes) or cell death (tumor suppressors). Oncogenic mutations, gene deletions or amplifications alter the expression and/or function of these proteins, thereby changing the delicate balance between oncogenes and tumor suppressors, impacting oncogenesis and favoring malignant cell function and behavior. In this review, we provided an integrated overview of the impact of the major oncogenes and tumor suppressors, often altered in cancer cells, on Ca(2+) signaling from the ER Ca(2+) stores. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

  12. Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNA(Tyr) Suppressor.

    PubMed

    Rauch, Benjamin J; Porter, Joseph J; Mehl, Ryan A; Perona, John J

    2016-01-26

    The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(Tyr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(Tyr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(Tyr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes. PMID:26694948

  13. A novel proapoptotic gene PANO encodes a post-translational modulator of the tumor suppressor p14ARF

    SciTech Connect

    Watari, Akihiro; Li, Yang; Higashiyama, Shinji; Yutsudo, Masuo

    2012-02-01

    The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene.

  14. Infrared suppressor effect on T63 turboshaft engine performance

    NASA Technical Reports Server (NTRS)

    Bailey, E. E.; Civinskas, K. C.; Walker, C. L.

    1978-01-01

    Tests were conducted to determine if there are performance penalties associated with the installation of infrared (IR) suppressors on the T63-A-700 turboshaft engine. The testing was done in a sea-level, static test cell. The same engine (A-E402808 B) was run with the standard OH-58 aircraft exhaust stacks and with the ejector-type IR suppressors in order to make a valid comparison. Repeatability of the test results for the two configurations was verified by rerunning the conditions over a period of days. Test results showed no measurable difference in performance between the standard exhaust stacks and the IR suppressors.

  15. Suppressor cells in Trypanosoma congolense-infected mice.

    PubMed

    Pearson, T W; Roelants, G E; Lundin, L B; Mayor-Withey, K S

    1979-01-01

    Spleen cells from mice infected with T. congolense strongly suppressed lymphocyte stimulation induced in normal spleen cells by incubation with mitogens or allogeneic cells. Cell dilution studies showed that suppressor activity was extremely strong. Suppressor cell activity was markedly reduced by treatment of spleen cell populations with mitomycin-C and was unaffected by treatment with anti-Thy.1 sera and complement. Removal of cells which bound carbonyl iron or which bound to nylon columns, decreased but did not abolish suppressor activity. PMID:313686

  16. Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue

    SciTech Connect

    Wallentine, Brad D.; Wang, Ying; Tretyachenko-Ladokhina, Vira; Tan, Martha; Senear, Donald F.; Luecke, Hartmut

    2013-10-01

    X-ray crystallographic structures of four p53 core-domain variants were determined in order to gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53. To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol{sup −1} (15.1 kJ mol{sup −1}). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the

  17. Drafting the proteome landscape of myeloid-derived suppressor cells.

    PubMed

    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. PMID:26403437

  18. Functional involvement of human discs large tumor suppressor in cytokinesis

    SciTech Connect

    Unno, Kenji; Hanada, Toshihiko; Chishti, Athar H.

    2008-10-15

    Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.

  19. Functional involvement of human discs large tumor suppressor in cytokinesis

    PubMed Central

    Unno, Kenji; Hanada, Toshihiko; Chishti, Athar H.

    2008-01-01

    Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis. PMID:18760273

  20. Tumor suppressor VHL functions in the control of mitotic fidelity.

    PubMed

    Hell, Michael P; Duda, Maria; Weber, Thomas C; Moch, Holger; Krek, Wilhelm

    2014-05-01

    The von Hippel-Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell-type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo.

  1. Tumor suppressor WWOX moderates the mitochondrial respiratory complex.

    PubMed

    Choo, Amanda; O'Keefe, Louise V; Lee, Cheng Shoou; Gregory, Stephen L; Shaukat, Zeeshan; Colella, Alexander; Lee, Kristie; Denton, Donna; Richards, Robert I

    2015-12-01

    Fragile site FRA16D exhibits DNA instability in cancer, resulting in diminished levels of protein from the WWOX gene that spans it. WWOX suppresses tumor growth by an undefined mechanism. WWOX participates in pathways involving aerobic metabolism and reactive oxygen species. WWOX comprises two WW domains as well as a short-chain dehydrogenase/reductase enzyme. Herein is described an in vivo genetic analysis in Drosophila melanogaster to identify functional interactions between WWOX and metabolic pathways. Altered WWOX levels modulate variable cellular outgrowths caused by genetic deficiencies of components of the mitochondrial respiratory complexes. This modulation requires the enzyme active site of WWOX, and the defective respiratory complex-induced cellular outgrowths are mediated by reactive oxygen species, dependent upon the Akt pathway and sensitive to levels of autophagy and hypoxia-inducible factor. WWOX is known to contribute to homeostasis by regulating the balance between oxidative phosphorylation and glycolysis. Reduction of WWOX levels results in diminished ability to respond to metabolic perturbation of normal cell growth. Thus, the ability of WWOX to facilitate escape from mitochondrial damage-induced glycolysis (Warburg effect) is, therefore, a plausible mechanism for its tumor suppressor activity.

  2. Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function

    SciTech Connect

    Yoshihara, Takashi; Collado, Denise; Hamaguchi, Masaaki . E-mail: hamaguchi@fordham.edu

    2007-07-13

    The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.

  3. SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis

    PubMed Central

    Ozturk, Sait; Papageorgis, Panagiotis; Wong, Chen Khuan; Lambert, Arthur W.; Abdolmaleky, Hamid M.; Thiagalingam, Arunthathi; Cohen, Herbert T.; Thiagalingam, Sam

    2016-01-01

    Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis–free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications. PMID:26739564

  4. RASSF10 is epigenetically silenced and functions as a tumor suppressor in gastric cancer

    SciTech Connect

    Wei, Ziran; Chen, Xia; Chen, Ji; Wang, Weimin; Xu, Xudong; Cai, Qingping

    2013-03-22

    Highlights: ► Epigenetic silencing of RASSF10 gene expression in GC cells. ► RASSF10 overexpression inhibits cell growth in vitro and in vivo. ► RASSF10 induces apoptosis in GC cells. ► RASSF10 inhibits Wnt/β-catenin signaling pathway. -- Abstract: Ras association domain family (RASSF) proteins are encoded by several tumor suppressor genes that are frequently silenced in human cancers. In this study, we investigated RASSF10 as a target of epigenetic inactivation and examined its functions as a tumor suppressor in gastric cancer. RASSF10 was silenced in six out of eight gastric cancer cell lines. Loss or downregulation of RASSF10 expression was associated with promoter hypermethylation, and could be restored by a demethylating agent. Overexpression of RASSF10 in gastric cancer cell lines (JRST, BGC823) suppressed cell growth and colony formation, and induced apoptosis, whereas RASSF10 depletion promoted cell growth. In xenograft animal experiments, RASSF10 overexpression effectively repressed tumor growth. Mechanistic investigations revealed that RASSF10 inhibited tumor growth by blocking activation of β-catenin and its downstream targets including c-Myc, cyclinD1, cyclinE1, peroxisome proliferator-activated receptor δ, transcription factor 4, transcription factor 1 and CD44. In conclusion, the results of this study provide insight into the role of RASSF10 as a novel functional tumor suppressor in gastric cancer through inhibition of the Wnt/β-catenin signaling pathway.

  5. Genetic characterization of frameshift suppressors with new decoding properties.

    PubMed Central

    Hughes, D; Thompson, S; O'Connor, M; Tuohy, T; Nichols, B P; Atkins, J F

    1989-01-01

    Suppressor mutants that cause ribosomes to shift reading frame at specific and new sequences are described. Suppressors for trpE91, the only known suppressible -1 frameshift mutant, have been isolated in Escherichia coli and in Salmonella typhimurium. E. coli hopR acts on trpE91 within the 9-base-pair sequence GGA GUG UGA, is dominant, and is located at min 52 on the chromosome. Its Salmonella homolog maps at an equivalent position and arises as a rarer class in that organism as compared with E. coli. The Salmonella suppressor, hopE, believed to be in a duplicate copy of the same gene, maps at min 17. The +1 suppressor, sufT, acts at the nonmonotonous sequence CCGU, is dominant, and maps at min 59 on the Salmonella chromosome. PMID:2644219

  6. SOCS1 in cancer: An oncogene and a tumor suppressor.

    PubMed

    Beaurivage, Claudia; Champagne, Audrey; Tobelaim, William S; Pomerleau, Véronique; Menendez, Alfredo; Saucier, Caroline

    2016-06-01

    The Suppressor Of Cytokine Signaling 1 (SOCS1) has been extensively investigated in immune cells where it works as a potent inhibitor of inflammation by negative feedback regulation of the cytokine-activated JAK-STAT signaling pathways. SOCS1 is also recognized as a tumor suppressor in numerous cancers and its critical functional relevance in non-immune cells, including epithelial cells, has just begun to emerge. Most notably, conflicting results from clinical and experimental studies suggest that SOCS1 may function as either a tumor suppressor or a tumor promoter, in a cell context-dependent manner. Here, we present an overview of the mechanisms underlying SOCS1 function as a tumor suppressor and discuss the emerging evidences of SOCS1 activity as an oncogene. PMID:26811119

  7. Cyclin C is a haploinsufficient tumor suppressor

    PubMed Central

    Li, Na; Fassl, Anne; Chick, Joel; Inuzuka, Hiroyuki; Li, Xiaoyu; Mansour, Marc R.; Liu, Lijun; Wang, Haizhen; King, Bryan; Shaik, Shavali; Gutierrez, Alejandro; Ordureau, Alban; Otto, Tobias; Kreslavsky, Taras; Baitsch, Lukas; Bury, Leah; Meyer, Clifford A.; Ke, Nan; Mulry, Kristin A.; Kluk, Michael J.; Roy, Moni; Kim, Sunkyu; Zhang, Xiaowu; Geng, Yan; Zagozdzon, Agnieszka; Jenkinson, Sarah; Gale, Rosemary E.; Linch, David C.; Zhao, Jean J.; Mullighan, Charles G.; Harper, J. Wade; Aster, Jon C.; Aifantis, Iannis; von Boehmer, Harald; Gygi, Steven P.; Wei, Wenyi; Look, A. Thomas; Sicinski, Piotr

    2014-01-01

    Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumor suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an “orphan” CDK19 kinase, as well as CDK8 and CDK3. These cyclin C-CDK complexes phosphorylate Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin C-knockout mice. Cyclin C ablation or heterozygosity collaborate with other oncogenic lesions and accelerate development of T-cell-acute lymphoblastic leukemia (T-ALL). Furthermore, the cyclin C gene is heterozygously deleted in a significant fraction of human T-ALL, and these tumors express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin C-CDK unable to phosphorylate ICN1. Hence, tumor cells may develop different strategies to evade cyclin C inhibitory function. PMID:25344755

  8. Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

    PubMed Central

    McClain, W H; Foss, K; Jenkins, R A; Schneider, J

    1990-01-01

    We have constructed an opal suppressor system in Escherichia coli to complement an existing amber suppressor system to study the structural basis of tRNA acceptor identity, particularly the role of middle anticodon nucleotide at position 35. The opal suppressor tRNA contains a UCA anticodon and the mRNA of the suppressed protein (which is easily purified and sequenced) contains a UGA nonsense triplet. Opal suppressor tRNAs of two tRNA(Arg) isoacceptor sequences each gave arginine in the suppressed protein, while the corresponding amber suppressors with U35 in their CUA anticodons each gave arginine plus a second amino acid in the suppressed protein. Since C35 but not U35 is present in the anticodon of wild-type tRNA(Arg) molecules, while the first anticodon position contains either C34 or U34, these results establish that C35 contributes to tRNA(Arg) acceptor identity. Initial characterizations of opal suppressor tRNA(Arg) mutants by suppression efficiency measurements suggest that the fourth nucleotide from the 3' end of tRNA(Arg) (A73 or G73 in different isoacceptors) also contributes to tRNA(Arg) acceptor identity. Wild-type and mutant versions of opal and amber tRNA(Lys) suppressors were examined, revealing that U35 and A73 are important determinants of tRNA(Lys) acceptor identity. Several possibilities are discussed for the general significance of having tRNA acceptor identity in the same positions in different tRNA acceptor types, as exemplified by positions 35 and 73 in tRNA(Arg) and tRNA(Lys). PMID:2251270

  9. Hampering the Immune Suppressors: Therapeutic Targeting of Myeloid-Derived Suppressor Cells (MDSC) in Cancer

    PubMed Central

    Albeituni, Sabrin Husein; Ding, Chuanlin; Yan, Jun

    2014-01-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive properties that preferentially expand in cancer. MDSC mainly suppress T cell proliferation and cytotoxicity, inhibit NK cell activation, and induce the differentiation and expansion of regulatory T cells (Tregs). The wide spectrum of MDSC suppressive activity in cancer and its role in tumor progression have rendered these cells a promising target for effective cancer immunotherapy. In this review we briefly discuss the origin of MDSC and their main mechanisms of suppression and focus more on the approaches developed up to date targeting of MDSC in tumor-bearing animals and cancer patients. PMID:24270348

  10. Simultaneous loss of the DLC1 and PTEN tumor suppressors enhances breast cancer cell migration

    SciTech Connect

    Heering, Johanna; Erlmann, Patrik; Olayioye, Monilola A.

    2009-09-10

    The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. The protein acts as a dual specificity phosphatase for lipids and proteins. PTEN loss confers a growth advantage to cells, protects from apoptosis and favors cell migration. The deleted in liver cancer 1 (DLC1) gene has emerged as a novel tumor suppressor downregulated in a variety of tumor types including those of the breast. DLC1 contains a Rho GTPase activating domain that is involved in the inhibition of cell proliferation, migration and invasion. To investigate how simultaneous loss of PTEN and DLC1 contributes to cell transformation, we downregulated both proteins by RNA interference in the non-invasive MCF7 breast carcinoma cell line. Joint depletion of PTEN and DLC1 resulted in enhanced cell migration in wounding and chemotactic transwell assays. Interestingly, both proteins were found to colocalize at the plasma membrane and interacted physically in biochemical pulldowns and coimmunoprecipitations. We therefore postulate that the concerted local inactivation of signaling pathways downstream of PTEN and DLC1, respectively, is required for the tight control of cell migration.

  11. Structural and functional characterization of the acidic region from the RIZ tumor suppressor.

    PubMed

    Sun, Yizhi; Stine, Jessica M; Atwater, Daniel Z; Sharmin, Ayesha; Ross, J B Alexander; Briknarová, Klára

    2015-02-17

    RIZ (retinoblastoma protein-interacting zinc finger protein), also denoted PRDM2, is a transcriptional regulator and tumor suppressor. It was initially identified because of its ability to interact with another well-established tumor suppressor, the retinoblastoma protein (Rb). A short motif, IRCDE, in the acidic region (AR) of RIZ was reported to play an important role in the interaction with the pocket domain of Rb. The IRCDE motif is similar to a consensus Rb-binding sequence LXCXE (where X denotes any amino acid) that is found in several viral Rb-inactivating oncoproteins. To improve our understanding of the molecular basis of binding of Rb to RIZ, we investigated the interaction between purified recombinant AR and the pocket domain of Rb using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy experiments. We show that AR is intrinsically disordered and that it binds the pocket domain with submicromolar affinity. We also demonstrate that the interaction between AR and the pocket domain is mediated primarily by the short stretch of residues containing the IRCDE motif and that the contribution of other parts of AR to the interaction with the pocket domain is minimal. Overall, our data provide clear evidence that RIZ is one of the few cellular proteins that can interact directly with the LXCXE-binding cleft on Rb.

  12. Structural and Functional Characterization of the Acidic Region from the RIZ Tumor Suppressor

    PubMed Central

    Sun, Yizhi; Stine, Jessica M.; Atwater, Daniel Z.; Sharmin, Ayesha; Ross, J. B. Alexander; Briknarová, Klára

    2015-01-01

    RIZ (retinoblastoma protein-interacting zinc finger protein), also denoted PRDM2, is a transcriptional regulator and tumor suppressor. It was initially identified because of its ability to interact with another well-established tumor suppressor, the retinoblastoma protein (Rb). A short motif, IRCDE, in the acidic region (AR) of RIZ was reported to play an important role in the interaction with the pocket domain of Rb. The IRCDE motif is similar to a consensus Rb-binding sequence LXCXE (where X denotes any amino acid) that is found in several viral Rb-inactivating oncoproteins. To improve our understanding of the molecular basis of binding of Rb to RIZ, we investigated the interaction between purified recombinant AR and the pocket domain of Rb using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy experiments. We show that AR is intrinsically disordered and that it binds the pocket domain with submicromolar affinity. We also demonstrate that the interaction between AR and the pocket domain is mediated primarily by the short stretch of residues containing the IRCDE motif and that the contribution of other parts of AR to the interaction with the pocket domain is minimal. Overall, our data provide clear evidence that RIZ is one of the few cellular proteins that can interact directly with the LXCXE-binding cleft on Rb. PMID:25640033

  13. Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 2: Evaluating suppression efficiency

    PubMed Central

    Rodriguez, Erik A.; Lester, Henry A.; Dougherty, Dennis A.

    2007-01-01

    The incorporation of unnatural amino acids into proteins is a valuable tool for addition of biophysical probes, bio-orthogonal functionalities, and photoreactive cross-linking agents, although these approaches often require quantities of protein that are difficult to access with chemically aminoacylated tRNAs. THG73 is an amber suppressor tRNA that has been used ex