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Sample records for protein bcl-xl negatively

  1. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    PubMed

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response. PMID:26245900

  2. Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

    PubMed Central

    Cimmino, Amelia; Capasso, Rosanna; Muller, Fabbri; Sambri, Irene; Masella, Lucia; Raimo, Marianna; De Bonis, Maria Luigia; D'Angelo, Stefania; Zappia, Vincenzo; Galletti, Patrizia; Ingrosso, Diego

    2008-01-01

    Background Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). Methodology/Principal Findings Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H2O2; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H2O2 concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H2O2 treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative

  3. Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL.

    PubMed

    Yao, Yong; Nisan, Danielle; Fujimoto, Lynn M; Antignani, Antonella; Barnes, Ashley; Tjandra, Nico; Youle, Richard J; Marassi, Francesca M

    2016-06-01

    BCL-XL is a dominant inhibitor of apoptosis and a significant anti-cancer drug target. Endogenous BCL-XL is integral to the mitochondrial outer membrane (MOM). BCL-XL reconstituted in detergent-free lipid bilayer nanodiscs is anchored to the nanodisc lipid bilayer membrane by tight association of its C-terminal tail, while the N-terminal head retains the canonical structure determined for water-soluble, tail-truncated BCL-XL, with the surface groove solvent-exposed and available for BH3 ligand binding. To better understand the conformation and dynamics of this key region of BCL-XL we have developed methods for isolating the membrane-embedded C-terminal tail from its N-terminal head and for preparing protein suitable for structural and biochemical studies. Here, we outline the methods for sample preparation and characterization and describe previously unreported structural and dynamics features. We show that the C-terminal tail of BCL-XL forms a transmembrane α-helix that retains a significant degree of conformational dynamics. We also show that the presence of the intact C-terminus destabilizes the soluble state of the protein, and that the small fraction of soluble recombinant protein produced in Escherichia coli is susceptible to proteolytic degradation of C-terminal residues beyond M218. This finding impacts the numerous previous studies where recombinant soluble BCL-XL was presumed to be full-length. Nevertheless, the majority of recombinant BCL-XL produced in E. coli is insoluble and protected from proteolysis. This protein retains the complete C-terminal tail and can be reconstituted in lipid bilayers in a folded and active state. PMID:26923059

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  5. Structural insights into mouse anti-apoptotic Bcl-xl reveal affinity for Beclin 1 and gossypol

    SciTech Connect

    Priyadarshi, Amit; Roy, Ankoor; Kim, Key-Sun; Kim, Eunice EunKyeong; Hwang, Kwang Yeon

    2010-04-09

    This study reports the crystal structures of Bcl-xl wild type and three Bcl-xl mutants (Y101A, F105A, and R139A) with amino acid substitutions in the hydrophobic groove of the Bcl-xl BH3 domain. An additional 12 ordered residues were observed in a highly flexible loop between the {alpha}1 and {alpha}2 helices, and were recognized as an important deamidation site for the regulation of apoptosis. The autophagy-effector protein, Beclin 1, contains a novel BH3 domain (residues 101-125), which binds to the surface cleft of Bcl-xl, as confirmed by nuclear magnetic resonance (NMR) spectroscopy and analytical gel-filtration results. Gossypol, a potent inhibitor of Bcl-xl, had a K{sub d} value of 0.9 {mu}M. In addition, the structural and biochemical analysis of five Bcl-xl substitution mutants will provide structural insights into the design and development of anti-cancer drugs.

  6. Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain

    PubMed Central

    Vervliet, Tim; Lemmens, Irma; Vandermarliere, Elien; Decrock, Elke; Ivanova, Hristina; Monaco, Giovanni; Sorrentino, Vincenzo; Kasri, Nael Nadif; Missiaen, Ludwig; Martens, Lennart; De Smedt, Humbert; Leybaert, Luc; Parys, Jan B.; Tavernier, Jan; Bultynck, Geert

    2015-01-01

    Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca2+-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca2+ release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XLK87D mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca2+ release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction. PMID:25872771

  7. Eliminating Legionella by inhibiting BCL-XL to induce macrophage apoptosis.

    PubMed

    Speir, Mary; Lawlor, Kate E; Glaser, Stefan P; Abraham, Gilu; Chow, Seong; Vogrin, Adam; Schulze, Keith E; Schuelein, Ralf; O'Reilly, Lorraine A; Mason, Kylie; Hartland, Elizabeth L; Lithgow, Trevor; Strasser, Andreas; Lessene, Guillaume; Huang, David C S; Vince, James E; Naderer, Thomas

    2016-01-01

    Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease(1). Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes. PMID:27572165

  8. Ectopic expression of Bcl-XL or Ku70 protects human colon cancer cells (SW480) against curcumin-induced apoptosis while their down-regulation potentiates it.

    PubMed

    Rashmi, Ramachandran; Kumar, Santhosh; Karunagaran, Devarajan

    2004-10-01

    Curcumin, the yellow pigment derived from Curcuma longa, is known to induce apoptosis of several cancer cells. However, many cancer cells protect themselves by over-expressing antiapoptotic proteins such as Bcl-XL or Ku70. To study their role in curcumin-induced apoptosis, human colon cancer cells (SW480) were made to over-express or under-express Bcl-XL (by stable transfection) and Ku70 (by transient transfection) using plasmid constructs that express their genes in sense or antisense orientation, respectively. Stable cells that express Bax [Bax-GFP (green fluorescent protein)], a proapoptotic member of the Bcl-2 family, were also established. Curcumin-induced cell death and nuclear condensation was more in AsBcl-XL and AsKu70 cells that under-express Bcl-XL and Ku70, respectively, compared with the vector-transfected cells. Bcl-XL and Ku70 protected the cells by inhibiting the release of cytochrome c, Smac (second mitochondria derived activator of caspase) and apoptosis inducing factor (AIF), and the activation of caspases 9, 8 and 3 triggered by curcumin. AsBcl-XL and AsKu70 cells were more sensitive to curcumin through enhanced activation of caspases 9 and 3 and release of cytochrome c, Smac and AIF. Curcumin-induced activation of caspase 8 was blocked by Ku70 but not by Bcl-XL. However, caspase 8 activation by curcumin was accelerated in both AsBcl-XL and AsKu70 cells suggesting a possible feedback activation of caspase 8 by caspase 3. Bax-GFP cells were highly sensitized when Ku70 was down-regulated supporting the reported role of Ku70 in the retention of Bax within the cytosol. The study reveals the potential of antisense inhibition of antiapoptotic proteins as an effective strategy to tackle chemoresistant cancers with curcumin. PMID:15205359

  9. Anti-apoptotic Bcl-XL but not Mcl-1 contributes to protection against virus-induced apoptosis.

    PubMed

    Ohmer, Michaela; Weber, Arnim; Sutter, Gerd; Ehrhardt, Katrin; Zimmermann, Albert; Häcker, Georg

    2016-01-01

    Infection of mammalian cells with viruses often induces apoptosis. How the recognition of viruses leads to apoptosis of the infected cell and which host cell factors regulate this cell death is incompletely understood. In this study, we focussed on two major anti-apoptotic proteins of the host cell, whose abundance and activity are important for cell survival, the Bcl-2-like proteins Mcl-1 and Bcl-XL. During infection of epithelial cells and fibroblasts with modified vaccinia virus Ankara (MVA), Mcl-1 protein levels dropped but the MVA Bcl-2-like protein F1L could replace Mcl-1 functionally; a similar activity was found in vaccinia virus (VACV)-infected cells. During infection with murine cytomegalovirus (MCMV), Mcl-1-levels were not reduced but a viral Mcl-1-like activity was also generated. Infection of mouse macrophages with any of these viruses, on the other hand, induced apoptosis. Virus-induced macrophage apoptosis was unaltered in the absence of Mcl-1. However, apoptosis was substantially increased in infected Bcl-XL-deficient macrophages or macrophages treated with the Bcl-2/Bcl-XL-inhibitor ABT-737. Genetic loss of Bcl-XL or treatment of macrophages with ABT-737 reduced the generation of infectious VACV. These data show that Mcl-1 is dispensable for the regulation of apoptosis during infection with different large DNA viruses, either because the viruses replace its function (in fibroblasts and epithelial cells) or because the pro-apoptotic activity generated by the infection appears not to be blocked by it (in macrophages). Bcl-XL, on the other hand, can be important to maintain survival of virus-infected cells, and its activity can determine outcome of the infection. PMID:27537523

  10. Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL

    PubMed Central

    Yosef, Reut; Pilpel, Noam; Tokarsky-Amiel, Ronit; Biran, Anat; Ovadya, Yossi; Cohen, Snir; Vadai, Ezra; Dassa, Liat; Shahar, Elisheva; Condiotti, Reba; Ben-Porath, Ittai; Krizhanovsky, Valery

    2016-01-01

    Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14ARF. Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies. PMID:27048913

  11. Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.

    PubMed

    Yosef, Reut; Pilpel, Noam; Tokarsky-Amiel, Ronit; Biran, Anat; Ovadya, Yossi; Cohen, Snir; Vadai, Ezra; Dassa, Liat; Shahar, Elisheva; Condiotti, Reba; Ben-Porath, Ittai; Krizhanovsky, Valery

    2016-01-01

    Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14(ARF). Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies. PMID:27048913

  12. High CIP2A levels correlate with an antiapoptotic phenotype that can be overcome by targeting BCL-XL in chronic myeloid leukemia.

    PubMed

    Lucas, C M; Milani, M; Butterworth, M; Carmell, N; Scott, L J; Clark, R E; Cohen, G M; Varadarajan, S

    2016-06-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a predictive biomarker of disease progression in many malignancies, including imatinib-treated chronic myeloid leukemia (CML). Although high CIP2A levels correlate with disease progression in CML, the underlying molecular mechanisms remain elusive. In a screen of diagnostic chronic phase samples from patients with high and low CIP2A protein levels, high CIP2A levels correlate with an antiapoptotic phenotype, characterized by downregulation of proapoptotic BCL-2 family members, including BIM, PUMA and HRK, and upregulation of the antiapoptotic protein BCL-XL. These results suggest that the poor prognosis of patients with high CIP2A levels is due to an antiapoptotic phenotype. Disrupting this antiapoptotic phenotype by inhibition of BCL-XL via RNA interference or A-1331852, a novel, potent and BCL-XL-selective inhibitor, resulted in extensive apoptosis either alone or in combination with imatinib, dasatinib or nilotinib, both in cell lines and in primary CD34(+) cells from patients with high levels of CIP2A. These results demonstrate that BCL-XL is the major antiapoptotic survival protein and may be a novel therapeutic target in CML. PMID:26987906

  13. High CIP2A levels correlate with an antiapoptotic phenotype that can be overcome by targeting BCL-XL in chronic myeloid leukemia

    PubMed Central

    Lucas, C M; Milani, M; Butterworth, M; Carmell, N; Scott, L J; Clark, R E; Cohen, G M; Varadarajan, S

    2016-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a predictive biomarker of disease progression in many malignancies, including imatinib-treated chronic myeloid leukemia (CML). Although high CIP2A levels correlate with disease progression in CML, the underlying molecular mechanisms remain elusive. In a screen of diagnostic chronic phase samples from patients with high and low CIP2A protein levels, high CIP2A levels correlate with an antiapoptotic phenotype, characterized by downregulation of proapoptotic BCL-2 family members, including BIM, PUMA and HRK, and upregulation of the antiapoptotic protein BCL-XL. These results suggest that the poor prognosis of patients with high CIP2A levels is due to an antiapoptotic phenotype. Disrupting this antiapoptotic phenotype by inhibition of BCL-XL via RNA interference or A-1331852, a novel, potent and BCL-XL-selective inhibitor, resulted in extensive apoptosis either alone or in combination with imatinib, dasatinib or nilotinib, both in cell lines and in primary CD34+ cells from patients with high levels of CIP2A. These results demonstrate that BCL-XL is the major antiapoptotic survival protein and may be a novel therapeutic target in CML. PMID:26987906

  14. Loss of PUMA (BBC3) does not prevent thrombocytopenia caused by the loss of BCL-XL (BCL2L1).

    PubMed

    Delbridge, Alex R D; Chappaz, Stephane; Ritchie, Matthew E; Kile, Benjamin T; Strasser, Andreas; Grabow, Stephanie

    2016-09-01

    Apoptosis is required to maintain tissue homeostasis in multicellular organisms. Platelets, the anucleate cells that are essential for blood clotting, are a prime example. Their brief life span in the circulation is regulated by the intrinsic apoptosis pathway. Pro-survival BCL-XL (also termed BCL2L1) is essential for platelet viability. It functions to restrain the pro-apoptotic BCL-2 family members BAK (also termed BAK1) and BAX, the essential mediators of intrinsic apoptosis. Genetic deletion or pharmacological inhibition of BCL-XL results in thrombocytopenia. Conversely, deletion of BAK in platelets doubles their circulating life span. However, what triggers platelet apoptosis in vivo remains unclear. The pro-apoptotic BH3-only proteins are essential for initiating apoptosis in nucleated cells, and there is some evidence to suggest they also play a role in platelet biology. We investigated whether PUMA (also termed BBC3), a potent BH3-only protein that can inhibit all pro-survival BCL-2 family members as well as directly activate BAX, regulates the death of platelets. Surprisingly, loss of PUMA had no impact on the loss of platelets caused by loss of BCL-XL. It therefore remains to be established whether other BH3-only proteins play a critical role in induction of apoptosis in platelets or whether their death is controlled solely by the interactions between BCL-XL with BAK and BAX. PMID:27221652

  15. Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

    PubMed Central

    Coen, Laurent; du Pasquier, David; Le Mevel, Sébastien; Brown, Spencer; Tata, Jamshed; Mazabraud, André; Demeneix, Barbara A.

    2001-01-01

    Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development. PMID:11427732

  16. Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

    PubMed

    Bhat, Vikas; Kurouski, Dmitry; Olenick, Max B; McDonald, Caleb B; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2012-12-01

    Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues. PMID:22960132

  17. Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

    SciTech Connect

    Dai, Guodong; Peng, Tao; Zhou, Xuhong; Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi; Yuan, Yulin

    2013-11-01

    Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

  18. Discovery of potent and selective benzothiazole hydrazone inhibitors of Bcl-XL.

    PubMed

    Sleebs, Brad E; Kersten, Wilhemus J A; Kulasegaram, Sanji; Nikolakopoulos, George; Hatzis, Effie; Moss, Rebecca M; Parisot, John P; Yang, Hong; Czabotar, Peter E; Fairlie, W Douglas; Lee, Erinna F; Adams, Jerry M; Chen, Lin; van Delft, Mark F; Lowes, Kym N; Wei, Andrew; Huang, David C S; Colman, Peter M; Street, Ian P; Baell, Jonathan B; Watson, Keith; Lessene, Guillaume

    2013-07-11

    Developing potent molecules that inhibit Bcl-2 family mediated apoptosis affords opportunities to treat cancers via reactivation of the cell death machinery. We describe the hit-to-lead development of selective Bcl-XL inhibitors originating from a high-throughput screening campaign. Small structural changes to the hit compound increased binding affinity more than 300-fold (to IC50 < 20 nM). This molecular series exhibits drug-like characteristics, low molecular weights (Mw < 450), and unprecedented selectivity for Bcl-XL. Surface plasmon resonance experiments afford strong evidence of binding affinity within the hydrophobic groove of Bcl-XL. Biological experiments using engineered Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on Bcl-XL for survival) and Bax/Bak deficient MEFs (insensitive to selective activation of Bcl-2-driven apoptosis) support a mechanism-based induction of apoptosis. This manuscript describes the first series of selective small-molecule inhibitors of Bcl-XL and provides promising leads for the development of efficacious therapeutics against solid tumors and chemoresistant cancer cell lines. PMID:23767404

  19. Radiosensitization by a novel Bcl-2 and Bcl-XL inhibitor S44563 in small-cell lung cancer.

    PubMed

    Loriot, Y; Mordant, P; Dugue, D; Geneste, O; Gombos, A; Opolon, P; Guegan, J; Perfettini, J-L; Pierre, A; Berthier, L K; Kroemer, G; Soria, J C; Depil, S; Deutsch, E

    2014-01-01

    Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-XL inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence. PMID:25232677

  20. In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

    PubMed

    Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

    2015-01-01

    Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak. PMID:26610208

  1. In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

    PubMed Central

    Senft, D; Weber, A; Saathoff, F; Berking, C; Heppt, M V; Kammerbauer, C; Rothenfusser, S; Kellner, S; Kurgyis, Z; Besch, R; Häcker, G

    2015-01-01

    Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak. PMID:26610208

  2. MCL-1, BCL-XL and MITF Are Diversely Employed in Adaptive Response of Melanoma Cells to Changes in Microenvironment

    PubMed Central

    Hartman, Mariusz L.; Talar, Beata; Gajos-Michniewicz, Anna; Czyz, Malgorzata

    2015-01-01

    Melanoma cells can switch their phenotypes in response to microenvironmental insults. Heterogeneous melanoma populations characterized by long-term growth and a high self-renewal capacity can be obtained in vitro in EGF(+)bFGF(+) medium whilst invasive potential of melanoma cells is increased in serum-containing cultures. In the present study, we have shown that originally these patient-derived melanoma populations exhibit variable expression of pro-survival genes from the BCL-2 family and inhibitors of apoptosis (IAPs), and differ in the baseline MCL-1 transcript stability as well. While being transferred to serum-containing medium, melanoma cells are well protected from death. Immediate adaptive response of melanoma cells selectively involves a temporary MCL-1 increase, both at mRNA and protein levels, and BCL-XL can complement MCL-1, especially in MITFlow populations. Thus, the extent of MCL-1 and BCL-XL contributions seems to be cell context-dependent. An increase in MCL-1 level results from a transiently enhanced stability of its transcript, but not from altered protein turnover. Inhibition of MCL-1 preceding transfer to serum-containing medium caused the induction of cell death in a subset of melanoma cells, which confirms the involvement of MCL-1 in melanoma cell survival during the rapid alteration of growth conditions. Additionally, immediate response to serum involves the transient increase in MITF expression and inhibition of ERK-1/2 activity. Uncovering the mechanisms of adaptive response to rapid changes in microenvironment may extend our knowledge on melanoma biology, especially at the stage of dissemination. PMID:26035829

  3. Targeting MCL-1/BCL-XL Forestalls the Acquisition of Resistance to ABT-199 in Acute Myeloid Leukemia

    PubMed Central

    Lin, Kevin H.; Winter, Peter S.; Xie, Abigail; Roth, Cullen; Martz, Colin A.; Stein, Elizabeth M.; Anderson, Grace R.; Tingley, Jennifer P.; Wood, Kris C.

    2016-01-01

    ABT-199, a potent and selective small-molecule antagonist of BCL-2, is being clinically vetted as pharmacotherapy for the treatment of acute myeloid leukemia (AML). However, given that prolonged monotherapy tends to beget resistance, we sought to investigate the means by which resistance to ABT-199 might arise in AML and the extent to which those mechanisms might be preempted. Here we used a pathway-activating genetic screen to nominate MCL-1 and BCL-XL as potential nodes of resistance. We then characterized a panel of ABT-199-resistant myeloid leukemia cell lines derived through chronic exposure to ABT-199 and found that acquired drug resistance is indeed driven by the upregulation of MCL-1 and BCL-XL. By targeting MCL-1 and BCL-XL, resistant AML cell lines could be resensitized to ABT-199. Further, preemptively targeting MCL-1 and/or BCL-XL alongside administration of ABT-199 was capable of delaying or forestalling the acquisition of drug resistance. Collectively, these data suggest that in AML, (1) the selection of initial therapy dynamically templates the landscape of acquired resistance via modulation of MCL-1/BCL-XL and (2) appropriate selection of initial therapy may delay or altogether forestall the acquisition of resistance to ABT-199. PMID:27283158

  4. Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65

    PubMed Central

    2013-01-01

    Background In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. Results The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. Conclusions The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm

  5. Sec6/8 regulates Bcl-2 and Mcl-1, but not Bcl-xl, in malignant peripheral nerve sheath tumor cells.

    PubMed

    Tanaka, Toshiaki; Kikuchi, Noriaki; Goto, Kaoru; Iino, Mitsuyoshi

    2016-05-01

    Sec6 and Sec8, which are components of the exocyst complex, has been concerned with various roles independent of its role in secretion, such as cell migration, invadopodia formation, cytokinesis, glucose uptake, and neural development. Given the vital roles of the exocyst complex in cellular and developmental processes, the disruption of its function may be closely related to various diseases such as cancer, diabetes, and neuronal disorders. Malignant peripheral nerve sheath tumors (MPNSTs) have high malignant potential and poor prognosis because of aggressive progression and metastasis. To date, no chemotherapeutic agents have been validated for MPNSTs treatment because how MPNSTs are resistant to chemotherapeutic agents remains unknown. This study demonstrates that combination of doxorubicin and sorafenib induces apoptosis in MPNST cells through downregulation of B cell lymphoma protein 2 (Bcl-2), Bcl-2-related protein long form of Bcl-x (Bcl-xl), and myeloid cell leukemia 1 (Mcl-1). Moreover, both Sec6 and Sec8 levels decreased after treatment with doxorubicin and sorafenib and were found to be associated with Bcl-2 and Mcl-1 expressions, but not Bcl-xl. Although Sec8 was found to be involved in the regulation of both Bcl-2 and Mcl-1 at the mRNA level, Sec6 regulated Bcl-2 at the mRNA level and the binding affinity of F-box and WD repeat domain containing 7 and Mcl-1, thereby controlling Mcl-1 at the protein level. Bcl-2 or Mcl-1 mRNA suppression by Sec6 or Sec8 depletion resulted in significant changes in nuclear factor-kappa B, cAMP response element, and p53 transcriptional activity. These results suggest that Sec6 and Sec8 are therapeutic target molecules in MPNST. PMID:26892009

  6. Acetaminophen induces a caspase-dependent and Bcl-XL sensitive apoptosis in human hepatoma cells and lymphocytes.

    PubMed

    Boulares, A Hamid; Zoltoski, Anna J; Stoica, Bogdan A; Cuvillier, Olivier; Smulson, Mark E

    2002-01-01

    Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity. PMID:12005112

  7. Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases.

    PubMed

    Carlage, Tyler; Kshirsagar, Rashmi; Zang, Li; Janakiraman, Vijay; Hincapie, Marina; Lyubarskaya, Yelena; Weiskopf, Andy; Hancock, William S

    2012-01-01

    Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance. PMID:22556165

  8. Effect of actual long-term spaceflight on BDNF, TrkB, p75, BAX and BCL-XL genes expression in mouse brain regions.

    PubMed

    Naumenko, V S; Kulikov, A V; Kondaurova, E M; Tsybko, A S; Kulikova, E A; Krasnov, I B; Shenkman, B S; Sychev, V N; Bazhenova, E Y; Sinyakova, N A; Popova, N K

    2015-01-22

    Mice of C57BL/6J strain were exposed to 1-month spaceflight on Russian biosatellite Bion-M1 to determine the effect of long-term actual spaceflight on the expression of genes involved in the processes of neurogenesis and apoptosis. Specifically, we focused on the genes encoding proapoptotic factor BAX, antiapoptotic factor BCL-XL, brain-derived neurotrophic factor (BDNF) and BDNF receptors TrkB and p75. Spaceflight reduced the expression of the antiapoptotic BCL-XL gene in the striatum and hypothalamus, but increased it in the hippocampus. To estimate environmental stress contribution into spaceflight effects we analyzed spaceflight-responsive genes in mice housed for 1 month on Earth in the same shuttle cabins that were used for spaceflight, and in mice of the laboratory control group. It was shown that 1-month shuttle cabin housing decreased BCL-XL gene expression in the striatum but failed to alter BCL-XL mRNA levels in the hippocampus or hypothalamus. Spaceflight failed to alter the expression of the proapoptotic BAX gene in all investigated brain structures, although the insignificant increase of the BAX mRNA level in the hippocampus of spaceflight mice was found. At the same time, shuttle cabin housing produced insignificant decrease in BAX gene expression in the hippocampus. In contrast to the BCL-XL gene, genes encoding BAX, BDNF as well as TrkB and p75 receptors did not respond to 30-day spaceflight. Thus, long-term spaceflight (1) did not affect the expression of genes encoding BDNF as well as TrkB and p75 receptors, (2) produced dysregulation in genetic control of the neuronal apoptosis, (3) implicated BCL-XL as the risk factor for spaceflight-induced behavioral abnormalities. PMID:25451288

  9. Silibilin-induces apoptosis in breast cancer cells by modulating p53, p21, Bak and Bcl-XL pathways.

    PubMed

    Pirouzpanah, Mohammad Bagher; Sabzichi, Mehdi; Pirouzpanah, Saeed; Chavoshi, Hadi; Samadi, Nasser

    2015-01-01

    Nowadays herbal-derived medicines are attracting attention as new sources of drugs with few side effects. Silibinin is a flavonoid compound with chemotheraputic effects on different cancers such as examples in the prostate, lung, colon and breast. In the present study, the cytotoxic effects of silibinin on MCF7 breast cancer cells were investigated. Apoptosis was determined by flow cytometry and the impact of silibinin on the expression of pivotal genes including Bak, P53, P21, BRCA1, BCL-X1 and ATM was analyzed. Treatment for 24h had a significant dose-dependent inhibitory effect on cell growth (p<0.05) with dose- and time- dependent induction of apoptosis (p<0.05). In addition, there were significant increases in BRCA1, ATM, Bak and Bcl-XL gene expression at the mRNA level with different concentrations of silibinin for 24 or 48 h (p<0.05). Taken together, the results suggest that silibinin inhibits the proliferation and induces apoptosis of MCF-7 cells by down-regulating Bak, P53, P21, BRCA1, BCL-Xl and thus may be considered as an effective adjuvant drug to produce a better chemopreventive response for the cancer therapy. PMID:25773855

  10. Tivantinib (ARQ 197) affects the apoptotic and proliferative machinery downstream of c-MET: role of Mcl-1, Bcl-xl and Cyclin B1

    PubMed Central

    Lu, Shuai; Török, Helga-Paula; Gallmeier, Eike; Kolligs, Frank T.; Rizzani, Antonia; Arena, Sabrina; Göke, Burkhard; Gerbes, Alexander L.; De Toni, Enrico N.

    2015-01-01

    Tivantinib, a c-MET inhibitor, is investigated as a second-line treatment of HCC. It was shown that c-MET overexpression predicts its efficacy. Therefore, a phase-3 trial of tivantinib has been initiated to recruit “c-MET-high”patients only. However, recent evidence indicates that the anticancer activity of tivantinib is not due to c-MET inhibition, suggesting that c-MET is a predictor of response to this compound rather than its actual target. By assessing the mechanisms underlying the anticancer properties of tivantinib we showed that this agent causes apoptosis and cell cycle arrest by inhibiting the anti-apoptotic molecules Mcl-1 and Bcl-xl, and by increasing Cyclin B1 expression regardless of c-MET status. However, we found that tivantinib might antagonize the antiapoptotic effects of c-MET activation since HGF enhanced the expression of Mcl-1 and Bcl-xl. In summary, we show that the activity of tivantinib is independent of c-MET and describe Mcl-1, Bcl-xl and Cyclin B1 as effectors of its antineoplastic effects in HCC cells. We suggest that the predictive effect of c-MET expression in part reflects the c-MET-driven overexpression of Mcl-1 and Bcl-xl in c-MET-high patients and that these molecules are considered as possible response predictors. PMID:26259250

  11. Crystal Structure of the Bcl-XL-Beclin 1 Peptide Complex

    SciTech Connect

    Oberstein,A.; Jeffrey, P.; Shi, Y.

    2007-01-01

    Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-X{sub L}. However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-X{sub L}. We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-X{sub L} at 2.5{angstrom} resolution. Reminiscent of previously determined Bcl-X{sub L}-BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-X{sub L}. These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.

  12. MAPK-mediated phosphorylation of GATA-1 promotes Bcl-XL expression and cell survival.

    PubMed

    Yu, Yung-Luen; Chiang, Yun-Jung; Chen, Yu-Chun; Papetti, Michael; Juo, Chiun-Gung; Skoultchi, Arthur I; Yen, Jeffrey J Y

    2005-08-19

    In the interleukin 3-dependent hematopoietic cell line Ba/F3, inhibition of mitogen-activated protein kinase, a member of the MAPK/c-Jun N-terminal kinase/stress-activated protein kinase kinase family that plays an important role in cell growth and death control, rapidly leads to severe apoptosis. However, most of the antiapoptotic substrates of MAPK remain to be identified. Here we report that, upon interleukin-3 stimulation of Ba/F3 cells, the transcription factor GATA-1 is strongly phosphorylated at residue serine 26 by a MAPK-dependent pathway. Phosphorylation of GATA-1 increases GATA-1-mediated transcription of the E4bp4 survival gene without significantly changing the DNA-binding affinity of GATA-1. Further characterization of GATA-1 phosphorylation site mutants revealed that the antiapoptotic function of GATA-1 is strongly dependent upon its phosphorylation at the Ser-26 position and is probably mediated through its up-regulation of Bcl-X(L) expression. Taken together, our data demonstrate that MAPK-dependent GATA-1 phosphorylation is important for its transactivation of the E4bp4 gene, Bcl-X(L) expression and cell survival. Therefore, GATA-1 may represent a novel MAPK substrate that plays an essential role in a cytokine-mediated antiapoptotic response. PMID:15967790

  13. RECOMBINANT BCL-XL ATTENUATES VASCULAR HYPERPERMEABILITY IN A RAT MODEL OF HEMORRHAGIC SHOCK

    PubMed Central

    Tharakan, Binu; McNeal, Sam I.; Hunter, Felicia A.; Sawant, Devendra A.; Smythe, W. Roy; Childs, Ed W.

    2016-01-01

    Following hemorrhagic shock (HS), vascular hyperpermeability i.e. the leakage of fluid, nutrients and proteins into the extravascular space occurs primarily due to the disruption of the endothelial cell-cell adherens junctional complex. Studies from our laboratory demonstrate that activation of the mitochondria mediated ‘intrinsic’ apoptotic signaling cascade has a significant role in modulating HS-induced hyperpermeability. Here we report the novel use of recombinant Bcl-xL, an anti-apoptotic protein, to control HS-induced vascular hyperpermeability. Our results corroborate involvement of vascular hyperpermeability and apoptotic signaling. Hemorrhagic shock (HS) (mean arterial pressure [MAP] was reduced to 40 mmHg for 60 minutes followed by resuscitation to 90 mmHg for 60 minutes) in rats resulted in vascular hyperpermeability as determined by intra-vital microscopy. Treatment of Bcl-xL (2.5ug/ml of rat blood in non-lipid cationic polymer, i.v.) before, during and even after HS attenuated or reversed HS-induced vascular hyperpermeability significantly (p<0.05). Conversely, treatment using Bcl-xL inhibitors, 2-methoxy antimycin (2-MeOAA) and ABT 737, significantly increased vascular hyperpermeability compared to sham (p<0.05). Bcl-xL treatment also decreased the amount of fluid volume required to maintain a MAP of 90 mmHg during resuscitation (p<0.05). HS resulted in increased mitochondrial ROS formation, reduction of ΔΨm, mitochondrial release of cytochrome c and significant activation of caspase-3 (p<0.05). All of these effects were significantly inhibited by Bcl-xL pre-treatment (p<0.05). Our results show that recombinant Bcl-xL is effective against HS-induced vascular hyperpermeability that appears to be mediated through preservation of ΔΨm and subsequent prevention of caspase-3 activation. PMID:27042339

  14. Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.

    PubMed

    Micali, Nicola; Ferrai, Carmelo; Fernandez-Diaz, Luis C; Blasi, Francesco; Crippa, Massimo P

    2009-03-01

    The Prep1 homeodomain transcription factor is essential in embryonic development. Prep1 hypomorphic mutant mouse (Prep1(i/i)) embryos (embryonic day 9.5) display an increased terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction compared to wild-type (WT) littermates. Prep1(i/i) mouse embryo fibroblasts (MEFs) show an increased basal level of annexin V binding activity, reduction of the mitochondrial-membrane potential, and increased caspase 9 and 3 activation, indicating increased apoptosis. Prep1(i/i) MEFs also respond faster than WT MEFs to genotoxic stress, indicating increased activation of the intrinsic apoptotic pathways. We did not observe an increase in p53 or an abnormal p53 response to apoptotic stimuli. However, hypomorphic MEFs have decreased endogenous levels of antiapoptotic Bcl-X(L) mRNA and protein, and Bcl-x overexpression rescues the defect of Prep1(i/i) MEFs. Using transient transfections and chromatin immunoprecipitation, we identified the Bcl-x promoter as a novel target of Prep1. Thus, Prep1 directly controls mitochondrial homeostasis (and the apoptotic potential) by modulating Bcl-x gene expression. PMID:19103748

  15. Expresssion of bax/bcl-xl by low-power laser irradiation in the Amyloid Beta 25-35 induced apoptosis of PC12 cell

    NASA Astrophysics Data System (ADS)

    Zhang, Lan; Xing, Da

    2006-09-01

    Apoptosis has been reported as a contributing pathophysiological mechanism of Alzheimer's disease (AD). Recently, the anti-apoptosis function of low-power laser irradiation (LPLI) was proposed, suggesting LPLI may become a new means for AD therapy. In this study, we aimed to demonstrate the anti-apoptosis function of LPLI at molecular level. Aβ 25-35 was used to induce apoptosis of PC12 cell, and then the cells were dealt with LPLI. After irradiation, the molecular level of apoptosis was detected by quantifying the bax I bcl-xl mRNA ratio using a highly sensitive and quantitative polymerase chain reaction (QT-PCR) technique. The primary results show that the bax Ibcl-xl mRNA ratio of the PC12 cell treated with Aβ 25-35 was decreased by LPLI, demonstrating the anti-apoptosis function of LPLI at molecular level.

  16. miR-491-5p-induced apoptosis in ovarian carcinoma depends on the direct inhibition of both BCL-XL and EGFR leading to BIM activation

    PubMed Central

    Denoyelle, C; Lambert, B; Meryet-Figuière, M; Vigneron, N; Brotin, E; Lecerf, C; Abeilard, E; Giffard, F; Louis, M-H; Gauduchon, P; Juin, P; Poulain, L

    2014-01-01

    We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-XL and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-XL expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations. PMID:25299770

  17. Functional antagonism between pro-apoptotic BIM and anti-apoptotic BCL-XL in MYC-induced lymphomagenesis.

    PubMed

    Delbridge, A R D; Grabow, S; Bouillet, P; Adams, J M; Strasser, A

    2015-04-01

    Genomic analyses revealed that many cancers have acquired abnormalities in their expression of pro- or anti-apoptotic members of the BCL-2 protein family. It is, however, unknown whether changes in pro- or anti-apoptotic BCL-2 family members have similar impact on tumorigenesis or whether changes in one subgroup have disproportionate impact. We compared the consequences of concomitant loss of anti-apoptotic Bclx and pro-apoptotic Bim on MYC-induced lymphomagenesis. Whereas only loss of both Bclx alleles markedly forestalled tumorigenesis, loss of a single Bim allele overcame this blockade. Conversely, loss of even a single Bim allele sufficed to substantially accelerate lymphomagenesis, and only loss of both but not loss of a single allele of Bclx could attenuate this acceleration. The evidence that modest (two-fold) monoallelic changes in the expression of at least some BH3-only proteins can profoundly impact tumorigenesis suggests that such aberrations, imposed by epigenetic or genetic changes, may expedite tumorigenesis more effectively than elevated expression of pro-survival BCL-2 family members. These findings further our understanding of the mechanisms of lymphomagenesis and possibly also cancer therapy. PMID:24858047

  18. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

    PubMed

    Bogenberger, J M; Kornblau, S M; Pierceall, W E; Lena, R; Chow, D; Shi, C-X; Mantei, J; Ahmann, G; Gonzales, I M; Choudhary, A; Valdez, R; Camoriano, J; Fauble, V; Tiedemann, R E; Qiu, Y H; Coombes, K R; Cardone, M; Braggio, E; Yin, H; Azorsa, D O; Mesa, R A; Stewart, A K; Tibes, R

    2014-08-01

    Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. PMID:24451410

  19. BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

    PubMed Central

    Bogenberger, J M; Kornblau, S M; Pierceall, W E; Lena, R; Chow, D; Shi, C-X; Mantei, J; Ahmann, G; Gonzales, I M; Choudhary, A; Valdez, R; Camoriano, J; Fauble, V; Tiedemann, R E; Qiu, Y H; Coombes, K R; Cardone, M; Braggio, E; Yin, H; Azorsa, D O; Mesa, R A; Stewart, A K; Tibes, R

    2014-01-01

    Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response. PMID:24451410

  20. Assessment of expressions of Bcl-XL, b-FGF, Bmp-2, Caspase-3, PDGFR-α, Smad1 and TGF-β1 genes in a rat model of lung ischemia/reperfusion

    PubMed Central

    Şimşek, Hasan; Demiryürek, Şeniz; Demir, Tuncer; Atabay, Hüsne Didem; Çeribasi, Ali Osman; Bayraktar, Recep; Kaplan, Davut Sinan; Öztuzcu, Serdar; Cengiz, Beyhan

    2016-01-01

    Objective(s): Ischemia is described as organs and tissues are destitute of oxygen due to decreased arterial or venous blood flow. Many mechanisms play role in cell death happened as a consequence of a new blood flow is needed for both cell regeneration and to clean toxic metabolites during ischemia and later. Lung damage induced by ischemia/reperfusion (I/R) is a frequent problem in lung transplantation. Apoptosis (programmed cell death) is known as cell suicide, and plays a key role in embryonic developmental and in maintain adult tissue’s life. Materials and Methods: It is investigated expressions of Smad1, Bmp-2, Bcl-XL, b-FGF, Caspase-3, TGF-β1, PDGFR-α genes for molecular changes in lung tissues, after I/R is formed, in this study. For this, we included 40 Wistar albino rats to this study and divided 4 groups (n=10). The Groups were determined as Control (C), Group 1= 1 hr ischemia (I), Group 2= 1 hr ischemia+2 hr reperfusion (I+2R), Group 3= 1 hr ischemia+4 hr reperfusion (I+4R). Besides, molecular analysis and histopathologic examinations of tissues were performed, and the results were evaluated by normalization and statistics analysis. Results: We have found a significant increase in expression of Bcl-XL (P=0.046) and Caspase-3 (P=0.026) genes of group 1, and it was not monitored any significant difference in Group 2 and Group 3. In all groups, the changes in b-FGF (P=0.087), Bmp-2 (P=0.457), TGF-β1 (P=0.201) and PDGFR-α (P=0.116) were not significant compared to control group. We did not see any mRNA expression of Smad1 gene in all groups include control. Conclusion: These findings suggest that I/R injury may trigger apoptotic mechanism in lung. PMID:27081467

  1. Acetogenins from Annona muricata as potential inhibitors of antiapoptotic proteins: a molecular modeling study.

    PubMed

    Antony, Priya; Vijayan, Ranjit

    2016-01-01

    Apoptosis is a highly regulated process crucial for maintaining cellular homeostasis and development. The B-cell lymphoma 2 (Bcl-2) family of proteins play a crucial role in regulating apoptosis. Overexpressed Bcl-2 proteins are associated with the development and progression of several human cancers. Annona muricata is a tropical plant that belongs to the Annonaceae family and is well known for its anticancer properties. In this study, molecular docking and simulations were performed to investigate the inhibitory potential of phytochemicals present in A. muricata against antiapoptotic proteins of the Bcl-2 family including Bcl-2, B-cell lymphoma extra-large (Bcl-Xl), and Mcl-1. Docking results revealed that the acetogenins, such as annomuricin A, annohexocin, muricatocin A, annomuricin-D-one, and muricatetrocin A/B, exhibited strong binding interactions with Bcl-Xl when compared to Bcl-2 and Mcl-1. Binding score and interactions of these acetogenins were notably better than those of currently available synthetic and natural inhibitors. Molecular dynamics simulations of the top-scoring lead molecules established that these molecules could bind strongly and consistently in the active site of Bcl-Xl. These results suggest that acetogenins could be explored as selective natural inhibitors of Bcl-Xl that could assist in promoting the intrinsic pathway of apoptosis. PMID:27110097

  2. Acetogenins from Annona muricata as potential inhibitors of antiapoptotic proteins: a molecular modeling study

    PubMed Central

    Antony, Priya; Vijayan, Ranjit

    2016-01-01

    Apoptosis is a highly regulated process crucial for maintaining cellular homeostasis and development. The B-cell lymphoma 2 (Bcl-2) family of proteins play a crucial role in regulating apoptosis. Overexpressed Bcl-2 proteins are associated with the development and progression of several human cancers. Annona muricata is a tropical plant that belongs to the Annonaceae family and is well known for its anticancer properties. In this study, molecular docking and simulations were performed to investigate the inhibitory potential of phytochemicals present in A. muricata against antiapoptotic proteins of the Bcl-2 family including Bcl-2, B-cell lymphoma extra-large (Bcl-Xl), and Mcl-1. Docking results revealed that the acetogenins, such as annomuricin A, annohexocin, muricatocin A, annomuricin-D-one, and muricatetrocin A/B, exhibited strong binding interactions with Bcl-Xl when compared to Bcl-2 and Mcl-1. Binding score and interactions of these acetogenins were notably better than those of currently available synthetic and natural inhibitors. Molecular dynamics simulations of the top-scoring lead molecules established that these molecules could bind strongly and consistently in the active site of Bcl-Xl. These results suggest that acetogenins could be explored as selective natural inhibitors of Bcl-Xl that could assist in promoting the intrinsic pathway of apoptosis. PMID:27110097

  3. Acid destabilization of the solution conformation of Bcl-XL does not drive its pH-dependent insertion into membranes

    PubMed Central

    Thuduppathy, Guruvasuthevan R.; Hill, R. Blake

    2006-01-01

    Regulation of programmed cell death by Bcl-xL is dependent on both its solution and integral membrane conformations. A conformational change from solution to membrane is also important in this regulation. This conformational change shows a pH-dependence similar to the translocation domain of diphtheria toxin, where an acid-induced molten globule conformation in the absence of lipid vesicles mediates the change from solution to membrane conformations. By contrast, Bcl-xLΔTM in the absence of lipid vesicles exhibits no gross conformational changes upon acidification as observed by near- and far-UV circular dichroism spectropolarimetry. Additionally, no significant local conformational changes upon acidification were observed by heteronuclear NMR spectroscopy of Bcl-xLΔTM. Under conditions that favor the solution conformation (pH 7.4), the free energy of folding for Bcl-xLΔTM (ΔG°) was determined to be 15.8 kcal·mol−1. Surprisingly, under conditions that favor a membrane conformation (pH 4.9), ΔG° was 14.6 kcal·mol−1. These results differ from those obtained with many other membrane-insertable proteins where acid-induced destabilization is important. Therefore, other contributions must be necessary to destabilize the solution conformation Bcl-xL and favor the membrane conformation at pH 4.9. Such contributions might include the presence of a negatively charged membrane or an electrostatic potential across the membrane. Thus, for proteins that adopt both solution and membrane conformations, an obligatory molten globule intermediate may not be necessary. The absence of a molten globule intermediate might have evolved to protect Bcl-xL from intracellular proteases as it undergoes this conformational change essential for its activity. PMID:16385002

  4. Genomic profiling of acquired resistance to apoptosis in cells derived from human atherosclerotic lesions: potential role of STATs, cyclinD1, BAD, and Bcl-XL.

    PubMed

    Gagarin, Dmitry; Yang, Zhaoqing; Butler, Jason; Wimmer, Monika; Du, Baoheng; Cahan, Patrick; McCaffrey, Timothy A

    2005-09-01

    Current theories suggest that atherosclerosis, plaque rupture, stroke, and restenosis after angioplasty may involve defective apoptotic mechanisms in vascular cells. Prior work has demonstrated that cells from human atherosclerotic lesions, and cells from the aorta of aged rats, exhibit functional resistance to apoptosis induced by TGF-beta and glucocorticoids. The present studies demonstrate that human lesion-derived cells (LDC) are also resistant to apoptosis induced by fas ligation compared to cells derived from the adjacent media, and that in vitro expansion of LDC causes acquired resistance to apoptosis. Microarray profiling of fas-resistant versus sensitive cells identified a set of genes including STATs, caspase 1, cyclin D1, Bcl-xL, VDAC2, and BAD. The STAT proteins have been implicated in resistance to apoptosis, potentially via their ability to modulate caspase 1 (ICE), Bcl-xL, and cyclin D1 expression. Western blot analysis of sensitive and resistant LDC clonal lines confirmed increases in cyclin D1, STAT6, Bcl-xL, and BAD, with decreased expression of caspase 1. Thus, transcript profiling has identified a potential pathway of apoptotic regulation in subsets of lesion cells. The resistant phenotype may contribute to plaque stability and excessive vascular repair, while sensitive cells may be involved in plaque rupture and infarction. The data suggests both genetic interventions and novel small-molecule inhibitors that may be effective modulators of apoptosis in atherosclerosis, angina, and in-stent restenosis. PMID:16005468

  5. Bcl-2 family proteins as targets for anticancer drug design.

    PubMed

    Huang, Z

    2000-12-27

    Bcl-2 family proteins are key regulators of programmed cell death or apoptosis that is implicated in many human diseases, particularly cancer. In recent years, they have attracted intensive interest in both basic research to understand the fundamental principles of cell survival and cell death and drug discovery to develop a new class of anticancer agents. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions in either inhibiting or promoting cell death. High expression of anti-apoptotic members such as Bcl-2 and Bcl-XL commonly found in human cancers contributes to neoplastic cell expansion and interferes with the therapeutic action of many chemotherapeutic drugs. The functional blockade of Bcl-2 or Bcl-XL could either restore the apoptotic process in tumor cells or sensitize these tumors for chemo- and radiotherapies. This article reviews the recent progress in the design and discovery of small molecules that block the anti-apoptotic function of Bcl-2 or Bcl-XL. These chemical inhibitors are effective modulators of apoptosis and promising leads for the further development of new anticancer agents. PMID:11426648

  6. Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids

    PubMed Central

    Kalinina, Tatyana S.; Bulygina, Veta V.; Lanshakov, Dmitry A.; Babluk, Ekaterina V.

    2015-01-01

    Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons. PMID:26624017

  7. Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.

    PubMed

    Xu, Mei; Liu, Chao; Zhou, Mi; Li, Qing; Wang, Renxiao; Kang, Jingwu

    2016-08-16

    A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors. PMID:27425825

  8. Hydrophobic surfactant proteins strongly induce negative curvature.

    PubMed

    Chavarha, Mariya; Loney, Ryan W; Rananavare, Shankar B; Hall, Stephen B

    2015-07-01

    The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote adsorption by inducing lipids to adopt a more negative curvature, closer to the configuration of the hypothetical intermediate. Our experiments used x-ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), the proteins produced a dose-related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With an increasing mol fraction of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w/w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Analysis of the results in terms of a Langmuir model for binding to a surface suggests that the effect of the lipids is consistent with a change in the maximum binding capacity. Our findings show that surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism. PMID:26153706

  9. Persea declinata (Bl.) Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation.

    PubMed

    Narrima, Putri; Paydar, Mohammadjavad; Looi, Chung Yeng; Wong, Yi Li; Taha, Hairin; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A Hamid A

    2014-01-01

    Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

  10. Persea declinata (Bl.) Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

    PubMed Central

    Wong, Yi Li; Wong, Won Fen; Ali Mohd, Mustafa; Hadi, A. Hamid A.

    2014-01-01

    Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development. PMID:24808916

  11. In vitro efficacy of AdTRAIL gene therapy of bladder cancer is enhanced by trichostatin A-mediated restoration of CAR expression and downregulation of cFLIP and Bcl-XL.

    PubMed

    El-Zawahry, A; Lu, P; White, S J; Voelkel-Johnson, C

    2006-03-01

    Current therapies for bladder cancer are suboptimal and adenoviral gene therapy has been explored as an alternative treatment. In this study, we evaluated the in vitro efficacy of an adenovirus expressing TNF-related apoptosis-inducing ligand (AdTRAIL). At low concentrations of virus, T24 cells were more resistant to AdTRAIL-induced apoptosis than 5637 bladder carcinoma cells. Resistance in T24 cells correlated with poor infectivity and lack of surface expression of coxsackie and adenovirus receptor (CAR). Pretreatment with low concentrations of the histone deacetylase inhibitor trichostatin A, restored CAR expression in T24 cells, which facilitated viral infection and resulted in apoptosis at low concentrations of AdTRAIL. In addition, trichostatin A reduced the expression of Bcl-X(L) and cFLIP resulting in increased sensitivity to recombinant TRAIL. Overexpression of cFLIP inhibited TRAIL-mediated killing in trichostatin A pretreated cells, indicating that downregulation of this antiapoptotic protein is required for sensitization. Therefore, trichostatin A can enhance the efficacy of AdTRAIL by restoring CAR expression and by generating a more pro-apoptotic phenotype that would facilitate bystander activity of TRAIL. Combination of histone deacetylase inhibitors with intravesical AdTRAIL gene therapy may be a novel treatment strategy for bladder cancer. PMID:16167063

  12. Increased expression of PcG protein YY1 negatively regulates B cell development while allowing accumulation of myeloid cells and LT-HSC cells.

    PubMed

    Pan, Xuan; Jones, Morgan; Jiang, Jie; Zaprazna, Kristina; Yu, Duonan; Pear, Warren; Maillard, Ivan; Atchison, Michael L

    2012-01-01

    Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs. PMID:22292011

  13. Epithelial cell expression of BCL-2 family proteins predicts mechanisms that regulate Helicobacter pylori-induced pathology in the mouse stomach

    PubMed Central

    Yang, David X; Tashima, Kimihito; Taylor, Nancy S; Fox, James G

    2009-01-01

    Corpus-predominant infection with Helicobacter pylori (HP) results in the activation of programmed cell death pathways in surface, parietal, and chief cells. At present, mechanisms that regulate these pathways to result in HP-associated pathology are not fully understood. Because it is not known which survival and death pathways are present in gastric epithelial cells, we used an antibody panel to evaluate the expression of BCL-2 family prosurvival proteins or multi-Bcl-2 homology (BH)-domains (group 1) or BH3-only (group-2) proapoptotic proteins in the stomachs of uninfected or HP-infected C57BL/6 mice. This strategy identified BCL-2, BAK, and BAD as the major prosurvival and proapoptotic proteins, in surface cells and BAD as the only BCL-2 family protein expressed in parietal cells. Chief cells express altogether different effectors, including BCL-XL/BCL-2, for survival but have no constitutively expressed proapoptotic proteins. In model chief cells, however, the group 1 proapoptotic protein BCL-XS was expressed after exposure to proinflammatory cytokines concomitant with reduced viability, demonstrating that chief cells can transcriptionally regulate the induction of proapoptotic proteins to execute apoptosis. During HP infection, no additional BCL-2 family proteins were expressed in epithelial cells, whereas those present either remained unchanged or were reduced as cell deletion occurred over time. Additional studies demonstrated that the posttranslational regulation of BAD in surface and parietal cells was negatively affected by HP infection, a result that may be directly related to an increase in apoptosis during infection. Thus, gastric epithelial cells express cell-specific prosurvival and proapoptotic pathways. From the results presented here, mechanisms that regulate HP-related changes in the survival and death profile of gastric epithelial cells can be predicted and then tested, with the ultimate goal of elucidating important therapeutic targets to

  14. Negative Ions Enhance Survival of Membrane Protein Complexes.

    PubMed

    Liko, Idlir; Hopper, Jonathan T S; Allison, Timothy M; Benesch, Justin L P; Robinson, Carol V

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein. Graphical Abstract ᅟ. PMID:27106602

  15. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  16. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-04-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  17. DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells.

    PubMed

    Pont, Lotte M E Berghauser; Naipal, Kishan; Kloezeman, Jenneke J; Venkatesan, Subramanian; van den Bent, Martin; van Gent, Dik C; Dirven, Clemens M F; Kanaar, Roland; Lamfers, Martine L M; Leenstra, Sieger

    2015-01-28

    HDAC inhibitors have radiosensitizing effects in established cancer cell lines. This study was conducted to compare the efficacy of SAHA, LBH589, Valproic Acid (VPA), MS275 and Scriptaid in the patient-derived glioblastoma model. In more detail, SAHA and LBH589 were evaluated to determine predictors of response. Acetylated-histone-H3, γH2AX/53BP1, (p)Chek2/ATM, Bcl-2/Bcl-XL, p21(CIP1/WAF1) and caspase-3/7 were studied in relation to response. SAHA sensitized 50% of cultures, LBH589 45%, VPA and Scriptaid 40% and MS275 60%. Differences after treatment with SAHA/RTx or LBH589/RTx in a sensitive and resistant culture were increased acetylated-H3, caspase-3/7 and prolonged DNA damage repair γH2AX/53BP1 foci. pChek2 was found to be associated with both SAHA/RTx and LBH589/RTx response with a positive predictive value (PPV) of 90%. Bcl-XL had a PPV of 100% for LBH589/RTx response. Incubation with HDACi 24 and 48 hours pre-RTx resulted in the best efficacy of combination treatment. In conclusion a subset of patient-derived glioblastoma cultures were sensitive to HDACi/RTx. For SAHA and LBH589 responses were strongly associated with pChek2 and Bcl-XL, which warrant further clinical exploration. Additional information on responsiveness was obtained by DNA damage response markers and apoptosis related proteins. PMID:25305451

  18. Metal nanoparticles triggered persistent negative photoconductivity in silk protein hydrogels

    NASA Astrophysics Data System (ADS)

    Gogurla, Narendar; Sinha, Arun K.; Naskar, Deboki; Kundu, Subhas C.; Ray, Samit K.

    2016-03-01

    Silk protein is a natural biopolymer with intriguing properties, which are attractive for next generation bio-integrated electronic and photonic devices. Here, we demonstrate the negative photoconductive response of Bombyx mori silk protein fibroin hydrogels, triggered by Au nanoparticles. The room temperature electrical conductivity of Au-silk hydrogels is found to be enhanced with the incorporation of Au nanoparticles over the control sample, due to the increased charge transporting networks within the hydrogel. Au-silk lateral photoconductor devices show a unique negative photoconductive response under an illumination of 325 nm, with excitation energy higher than the characteristic metal plasmon resonance band. The enhanced photoconductance yield in the hydrogels over the silk protein is attributed to the photo-oxidation of amino groups in the β-pleated sheets of the silk around the Au nanoparticles followed by the breaking of charge transport networks. The Au-silk nanocomposite does not show any photoresponse under visible illumination because of the localization of excited charges in Au nanoparticles. The negative photoconductive response of hybrid Au-silk under UV illumination may pave the way towards the utilization of silk for future bio-photonic devices using metal nanoparticle platforms.

  19. The rheostat in the membrane: BCL-2 family proteins and apoptosis

    PubMed Central

    Volkmann, N; Marassi, F M; Newmeyer, D D; Hanein, D

    2014-01-01

    Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that a balance between these opposing proteins, like a simple ‘rheostat', could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic–MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic stimuli. Thus, we can view the MOM as the central locale of a dynamic life–death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK–membrane assemblies. Future studies need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and function of the full-length proteins in membranes are needed to illuminate some of these critical issues. PMID

  20. [Protein a production in coagulase-negative or clumping factor (CF)-negative isolates of Staphylococcus aureus].

    PubMed

    Piechowicz, Lidia; Garbacz, Katarzyna; Wisniewska, Katarzyna; Dajnowska-Stanczewa, Agata; Galiński, Janusz

    2005-01-01

    The aim of study was to determine a production of proteinA in coagulase-negative Staphylococcus aureus (CNSA) or CF-negative S. aureus (CFNSA) strains. 59 CNSA and 18 CFNSA strains were isolated between 1997 and 2003 from different clinical specimens. The Protein A production was determined by immunoblotting method. The presence of protein A gene (spa) was investigated using the polymerase chain reaction (PCR). Two sets of phages and RFLP (Restriction Fragment Lenth Polymorphism) of coa gene method were used for typing strains. The results proved that the lack of ability of protein A production occurs more frequently in protein A-negative CFNSA strains with compare to the CNSA, which are protein A-positive for the majority of strains. Deficiencies of protein A, doesn't seem to be caused by the loss of spa gene. Protein A-negative CFNSA strains have phagotypes, RFLP and antibiotic resistant patterns which differ them from protein A-negative CNSA strains. Almost all of protein A-negative CFNSA and CNSA strains are resistant to methicillin. PMID:16494201

  1. Arabinogalactan proteins are incorporated in negatively charged coffee brew melanoidins.

    PubMed

    Bekedam, E Koen; De Laat, Marieke P F C; Schols, Henk A; Van Boekel, Martinus A J S; Smit, Gerrit

    2007-02-01

    The charge properties of melanoidins in high molecular weight (HMw) coffee brew fractions, isolated by diafiltration and membrane dialysis, were studied. Ion exchange chromatography experiments with the HMw fractions showed that coffee brew melanoidins were negatively charged whereas these molecules did not expose any positive charge at the pH of coffee brew. Fractions with different ionic charges were isolated and subsequently characterized by means of the specific extinction coefficient (K(mix 405nm)), sugar composition, phenolic group content, nitrogen content, and the arabinogalactan protein (AGP) specific Yariv gel-diffusion assay. The isolated fractions were different in composition and AGP was found to be present in one of the HMw fractions. The AGP accounted for 6% of the coffee brew dry matter and had a moderate negative charge, probably caused by the presence of uronic acids. As the fraction that precipitated with Yariv was brown (K(mix 405nm) = 1.2), compared to a white color in the green bean, it was concluded that these AGPs had undergone Maillard reaction resulting in an AGP-melanoidin complex. The presence of mannose (presumably from galactomannan) indicates the incorporation of galactomannans in the AGP-melanoidin complex. As the uronic acid content in the more negatively charged melanoidin-rich, AGP-poor HMw fractions decreased, it was hypothesized that acidic groups are formed or incorporated during melanoidin formation. PMID:17263472

  2. Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

    PubMed

    Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

    2014-03-15

    Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. PMID:24582986

  3. Choosing negative examples for the prediction of protein-protein interactions

    PubMed Central

    Ben-Hur, Asa; Noble, William Stafford

    2006-01-01

    The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions. PMID:16723005

  4. Ribosomal protein S14 negatively regulates c-Myc activity.

    PubMed

    Zhou, Xiang; Hao, Qian; Liao, Jun-Ming; Liao, Peng; Lu, Hua

    2013-07-26

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  5. A general system for studying protein-protein interactions in gram-negative bacteria

    SciTech Connect

    Pelletier, Dale A.; Hurst, G. B.; Foote, Linda J.; Lankford, Patricia K.; McKeown, Cathy K.; Lu, Tse-Yuan S.; Schmoyer, Denise D.; Shah, Manesh B.; Hervey IV, W. J.; McDonald, W. Hayes; Hooker, Brian S.; Cannon, William R.; Daly, Don S.; Gilmore, Jason M.; Wiley, H. S.; Auberry, Deanna L.; Wang, Yisong; Larimer, Frank; Kennel, S. J.; Doktycz, M. J.; Morrell-Falvey, Jennifer; Owens, Elizabeth T.; Buchanan, M. V.

    2008-08-01

    One of the most promising of the emerging methods for large-scale studies of interactions among proteins is co-isolation of an affinity-tagged protein and its interaction partners, followed by mass spectrometric identification of the co-purifying proteins. We describe a methodology for systematically identifying the proteins that interact with affinity-tagged “bait” proteins expressed from a medium copy plasmid, which are based on a broad host range (pBBR1MCS5) vector backbone that has been modified to incorporate the Gateway DEST plasmid multiple cloning region. This construct was designed to facilitate expression of fusion proteins bearing an affinity tag, across a range of Gram negative bacterial hosts. We demonstrate the performance of this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results from the RNA polymerase complex from these two species compared favorably with those for both plasmid- and chromosomally-encoded affinity-tagged fusion proteins expressed in a model organism, E. coli.

  6. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    PubMed Central

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  7. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  8. Korean Red Ginseng protects endothelial cells from serum-deprived apoptosis by regulating Bcl-2 family protein dynamics and caspase S-nitrosylation

    PubMed Central

    Kim, Young-Mi; Kim, Jung Hwan; Kwon, Hyuk Min; Lee, Dong Heon; Won, Moo-Ho; Kwon, Young-Guen; Kim, Young-Myeong

    2013-01-01

    Korean Red Ginseng extract (KRGE) is a traditional herbal medicine utilized to prevent endothelium dysfunction in the cardiovascular system; however, its underlying mechanism has not been clearly elucidated. We here examined the pharmacological effect and molecular mechanism of KRGE on apoptosis of human umbilical vein endothelial cells (HUVECs) in a serum-deprived apoptosis model. KRGE protected HUVECs from serum-deprived apoptosis by inhibiting mitochondrial cytochrome c release and caspase-9/-3 activation. This protective effect was significantly higher than that of American ginseng extract. KRGE treatment increased antiapoptotic Bcl-2 and Bcl-XL protein expression and Akt-dependent Bad phosphorylation. Moreover, KRGE prevented serum deprivation-induced subcellular redistribution of these proteins between the mitochondrion and the cytosol, resulting in suppression of mitochondrial cytochrome c release. In addition, KRGE increased nitric oxide (NO) production via Akt-dependent activation of endothelial NO synthase (eNOS), as well as inhibited caspase-9/-3 activities. These increases were reversed by co-treatment of cells with inhibitors of eNOS and phosphoinositide 3-kinase (PI3K) and pre-incubation of cell lysates in dithiothreitol, indicating KRGE induces NO-mediated caspase modification. Indeed, KRGE inhibited caspase-3 activity via S-nitrosylation. These findings suggest that KRGE prevents serum deprivation-induced HUVEC apoptosis via increased Bcl-2 and Bcl-XL protein expression, PI3K/Akt-dependent Bad phosphorylation, and eNOS/NO-mediated S-nitrosylation of caspases. The cytoprotective property of KRGE may be valuable for developing new pharmaceutical means that limit endothelial cell death induced during the pathogenesis of vascular diseases. PMID:24233159

  9. Direct induction of apoptosis using an optimal mitochondrially targeted p53.

    PubMed

    Mossalam, Mohanad; Matissek, Karina J; Okal, Abood; Constance, Jonathan E; Lim, Carol S

    2012-05-01

    Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-α (an inhibitor of p53 transcriptional activity), pifithrin-μ (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL

  10. DIRECT INDUCTION OF APOPTOSIS USING AN OPTIMAL MITOCHONDRIALLY TARGETED P53

    PubMed Central

    Mossalam, Mohanad; Matissek, Karina J.; Okal, Abood; Constance, Jonathan E.; Lim, Carol S.

    2012-01-01

    Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53.1, 2 p53 forms a complex with the anti-apoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization.3, 4 Although p53 performs its main role in the mitochondrial outer membrane it also interacts with different proteins in the mitochondrial inner membrane and matrix.5, 6 To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane (“XL-MTS” from Bcl-XL; “TOM-MTS” from TOM20), the inner membrane (“CCO-MTS” from cytochrome c oxidase) or matrix (“OTC-MTS” from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a non-toxic protein) as negative controls. We performed caspase-9, TUNEL, Annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its non-toxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-α (an inhibitor of p53 transcriptional activity), pifithrin-μ (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and over-expressing the anti-apoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through p53/Bcl-XL

  11. Nuclear NF-κB p65 phosphorylation at Serine 276 by protein kinase A contributes to the malignant phenotype of head and neck cancer

    PubMed Central

    Arun, Pattatheyil; Brown, Matthew; Ehsanian, Reza; Chen, Zhong; Van Waes, Carter

    2009-01-01

    Purpose Aberrant nuclear activation and phosphorylation of the canonical NF-κB subunit RELA/p65 at Serine-536 by Inhibitor Kappa B Kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-κB activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by Protein Kinase A (PKA) in the malignant phenotype, gene transactivation, and as a potential target for therapy. Experimental Design Phospho and total p65 protein expression and localization was determined in HNSCC tissue array and in cell lines. The effects of PKA inhibitor H-89 on cell proliferation and cell cycle and of H-89 and PKA specific siRNA knockdown on NF-κB activation and downstream gene expression were examined. Results Nuclear NF-κB p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, TNF-α significantly increased while H-89 inhibited constitutive and TNF-α induced nuclear p65-Ser276 phosphorylation, and significantly suppressed NF-κB and target gene IL-8 reporter activity. Knock down of PKA by siRNA inhibited NF-κB, IL-8 and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death and blocked the cell cycle in G1/S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-κB related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21CIP1/WAF1, while suppressing proliferative marker Ki67. Conclusions NF-κB RELA Ser276 phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC. PMID:19789307

  12. Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

    PubMed Central

    Booth, David S.; Avila-Sakar, Agustin; Cheng, Yifan

    2011-01-01

    Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation. In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users. PMID:22215030

  13. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders. PMID:26975317

  14. Negative activation enthalpies in the kinetics of protein folding.

    PubMed

    Oliveberg, M; Tan, Y J; Fersht, A R

    1995-09-12

    Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior. PMID:7568045

  15. Improving compound–protein interaction prediction by building up highly credible negative samples

    PubMed Central

    Liu, Hui; Sun, Jianjiang; Guan, Jihong; Zheng, Jie; Zhou, Shuigeng

    2015-01-01

    Motivation: Computational prediction of compound–protein interactions (CPIs) is of great importance for drug design and development, as genome-scale experimental validation of CPIs is not only time-consuming but also prohibitively expensive. With the availability of an increasing number of validated interactions, the performance of computational prediction approaches is severely impended by the lack of reliable negative CPI samples. A systematic method of screening reliable negative sample becomes critical to improving the performance of in silico prediction methods. Results: This article aims at building up a set of highly credible negative samples of CPIs via an in silico screening method. As most existing computational models assume that similar compounds are likely to interact with similar target proteins and achieve remarkable performance, it is rational to identify potential negative samples based on the converse negative proposition that the proteins dissimilar to every known/predicted target of a compound are not much likely to be targeted by the compound and vice versa. We integrated various resources, including chemical structures, chemical expression profiles and side effects of compounds, amino acid sequences, protein–protein interaction network and functional annotations of proteins, into a systematic screening framework. We first tested the screened negative samples on six classical classifiers, and all these classifiers achieved remarkably higher performance on our negative samples than on randomly generated negative samples for both human and Caenorhabditis elegans. We then verified the negative samples on three existing prediction models, including bipartite local model, Gaussian kernel profile and Bayesian matrix factorization, and found that the performances of these models are also significantly improved on the screened negative samples. Moreover, we validated the screened negative samples on a drug bioactivity dataset. Finally, we derived

  16. Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

    PubMed

    Amako, Yutaka; Syed, Gulam H; Siddiqui, Aleem

    2011-04-01

    Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process. PMID:21285358

  17. Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

    PubMed Central

    Tang, Z; Coleman, T R; Dunphy, W G

    1993-01-01

    The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase. Images PMID:7504624

  18. Negative example selection for protein function prediction: the NoGO database.

    PubMed

    Youngs, Noah; Penfold-Brown, Duncan; Bonneau, Richard; Shasha, Dennis

    2014-06-01

    Negative examples - genes that are known not to carry out a given protein function - are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html). PMID:24922051

  19. Negative Example Selection for Protein Function Prediction: The NoGO Database

    PubMed Central

    Youngs, Noah; Penfold-Brown, Duncan; Bonneau, Richard; Shasha, Dennis

    2014-01-01

    Negative examples – genes that are known not to carry out a given protein function – are rarely recorded in genome and proteome annotation databases, such as the Gene Ontology database. Negative examples are required, however, for several of the most powerful machine learning methods for integrative protein function prediction. Most protein function prediction efforts have relied on a variety of heuristics for the choice of negative examples. Determining the accuracy of methods for negative example prediction is itself a non-trivial task, given that the Open World Assumption as applied to gene annotations rules out many traditional validation metrics. We present a rigorous comparison of these heuristics, utilizing a temporal holdout, and a novel evaluation strategy for negative examples. We add to this comparison several algorithms adapted from Positive-Unlabeled learning scenarios in text-classification, which are the current state of the art methods for generating negative examples in low-density annotation contexts. Lastly, we present two novel algorithms of our own construction, one based on empirical conditional probability, and the other using topic modeling applied to genes and annotations. We demonstrate that our algorithms achieve significantly fewer incorrect negative example predictions than the current state of the art, using multiple benchmarks covering multiple organisms. Our methods may be applied to generate negative examples for any type of method that deals with protein function, and to this end we provide a database of negative examples in several well-studied organisms, for general use (The NoGO database, available at: bonneaulab.bio.nyu.edu/nogo.html). PMID:24922051

  20. Protein PEGylation attenuates adsorption and aggregation on a negatively charged and moderately hydrophobic polymer surface.

    PubMed

    Pai, Sheetal S; Przybycien, Todd M; Tilton, Robert D

    2010-12-01

    Covalent grafting of poly(ethylene glycol) chains to proteins ("PEGylation") is emerging as an effective technique to increase the in vivo circulation time and efficacy of protein drugs. PEGylated protein adsorption at a variety of solid/aqueous interfaces is a critical aspect of their manufacture, storage, and delivery. A special category of block copolymer, PEGylated proteins have one or more water-soluble linear polymer (PEG) blocks and a single globular protein block that each exert distinct intermolecular and surface interaction forces. We report the impact of PEGylation on protein adsorption at the interface between aqueous solutions and solid films of poly(lactide-co-glycolide) (PLG), a moderately hydrophobic and negatively charged polymer. Using the model protein lysozyme with controlled degrees of PEGylation, we employ total internal reflection fluorescence techniques to measure adsorption isotherms, adsorption reversibility, and the extent of surface-induced aggregation. Lysozyme PEGylation reduces the extent of protein adsorption and surface-induced aggregation and increases the reversibility of adsorption compared to the unconjugated protein. Results are interpreted in terms of steric forces among grafted PEG chains and their effects on protein-protein interactions and protein orientation on the surface. PMID:21067142

  1. Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection.

    PubMed

    Wang, Xu; Hwang, Sun-Young; Cong, Wei-Tao; Jin, Li-Tai; Choi, Jung-Kap

    2016-10-01

    In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches. PMID:27430933

  2. Protein domain networks: Scale-free mixing of positive and negative exponents

    NASA Astrophysics Data System (ADS)

    Nacher, J. C.; Hayashida, M.; Akutsu, T.

    2006-07-01

    Many biological studies have been focused on the study of proteins, since proteins are essential for most cell functions. Although proteins are unique, they share certain common properties. For example, well-defined regions within a protein can fold independently from the rest of the protein and have their own function. They are called protein domains, and served as protein building blocks. In this article, we present a theoretical model for studying the protein domain networks, where one node of the network corresponds to one protein and two proteins are connected if they contain the same domain. The resulting distribution of nodes with a given degree, k, shows not only a power-law with negative exponent γ=-1, but it resembles the superposition of two power-law functions, one with a negative exponent and another with a positive exponent β=1. We call this distribution pattern “ scale-free mixing”. To explain the emergence of this superposition of power-laws, we propose a basic model with two main components: (1) mutation and (2) duplication of domains. Precisely, duplication gives rise to complete subgraphs (i.e., cliques) on the network, thus for several values of k a large number of nodes with degree k is produced, which explains the positive power-law branch of the degree distribution. In order to compare our model with experimental data, we generate protein domain networks with data from the UniProt Knowledgebase-Swissprot database for protein sequences and using InterPro, Pfam and Smart for domain databases. Our results indicate that the signal of this scale-free mixing pattern is also observed in the experimental data and it is conserved among organisms as Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens.

  3. The Ubiquitin-like Protein PLIC-2 Is a Negative Regulator of G Protein-coupled Receptor Endocytosis

    PubMed Central

    N'Diaye, Elsa-Noah; Hanyaloglu, Aylin C.; Kajihara, Kimberly K.; Puthenveedu, Manojkumar A.; Wu, Ping; von Zastrow, Mark

    2008-01-01

    The activity of many signaling receptors is regulated by their endocytosis via clathrin-coated pits (CCPs). For G protein-coupled receptors (GPCRs), recruitment of the adaptor protein arrestin to activated receptors is thought to be sufficient to drive GPCR clustering in CCPs and subsequent endocytosis. We have identified an unprecedented role for the ubiquitin-like protein PLIC-2 as a negative regulator of GPCR endocytosis. Protein Linking IAP to Cytoskeleton (PLIC)-2 overexpression delayed ligand-induced endocytosis of two GPCRs: the V2 vasopressin receptor and β-2 adrenergic receptor, without affecting endocytosis of the transferrin or epidermal growth factor receptor. The closely related isoform PLIC-1 did not affect receptor endocytosis. PLIC-2 specifically inhibited GPCR concentration in CCPs, without affecting membrane recruitment of arrestin-3 to activated receptors or its cellular levels. Depletion of cellular PLIC-2 accelerated GPCR endocytosis, confirming its regulatory function at endogenous levels. The ubiquitin-like domain of PLIC-2, a ligand for ubiquitin-interacting motifs (UIMs), was required for endocytic inhibition. Interestingly, the UIM-containing endocytic adaptors epidermal growth factor receptor protein substrate 15 and Epsin exhibited preferential binding to PLIC-2 over PLIC-1. This differential interaction may underlie PLIC-2 specific effect on GPCR endocytosis. Identification of a negative regulator of GPCR clustering reveals a new function of ubiquitin-like proteins and highlights a cellular requirement for exquisite regulation of receptor dynamics. PMID:18199683

  4. Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases

    PubMed Central

    Ishida, A; Sueyoshi, N; Shigeri, Y; Kameshita, I

    2008-01-01

    Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their ‘switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their ‘switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases. PMID:18454172

  5. A novel pathway for outer membrane protein biogenesis in Gram‐negative bacteria

    PubMed Central

    Jeeves, Mark

    2015-01-01

    Summary The understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM. PMID:26059329

  6. A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria.

    PubMed

    Jeeves, Mark; Knowles, Timothy J

    2015-08-01

    The understanding of the biogenesis of the outer membrane of Gram-negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β-barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram-negative outer membrane that is independent of the BAM complex and TAM. PMID:26059329

  7. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    SciTech Connect

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  8. Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

    PubMed Central

    Kramer, Ryan M.; Shende, Varad R.; Motl, Nicole; Pace, C. Nick; Scholtz, J. Martin

    2012-01-01

    Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. PMID:22768947

  9. Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

    PubMed Central

    Falb, D; Maniatis, T

    1992-01-01

    Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors. Images PMID:1508206

  10. MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors

    PubMed Central

    Hiraki, Masayuki; Suzuki, Yozo; Alam, Maroof; Hinohara, Kunihiko; Hasegawa, Masanori; Jin, Caining; Kharbanda, Surender; Kufe, Donald

    2016-01-01

    Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC. PMID:27217294

  11. MUC1-C Stabilizes MCL-1 in the Oxidative Stress Response of Triple-Negative Breast Cancer Cells to BCL-2 Inhibitors.

    PubMed

    Hiraki, Masayuki; Suzuki, Yozo; Alam, Maroof; Hinohara, Kunihiko; Hasegawa, Masanori; Jin, Caining; Kharbanda, Surender; Kufe, Donald

    2016-01-01

    Aberrant expression of myeloid cell leukemia-1 (MCL-1) is a major cause of drug resistance in triple-negative breast cancer (TNBC) cells. Mucin 1 (MUC1) is a heterodimeric oncoprotein that is aberrantly overexpressed in most TNBC. The present studies show that targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in TNBC cells with silencing or pharmacologic inhibition with GO-203 is associated with downregulation of MCL-1 levels. Targeting MUC1-C suppresses the MEK → ERK and PI3K → AKT pathways, and in turn destabilizes MCL-1. The small molecules ABT-737 and ABT-263 target BCL-2, BCL-XL and BCL-w, but not MCL-1. We show that treatment with ABT-737 increases reactive oxygen species and thereby MUC1-C expression. In this way, MUC1-C is upregulated in TNBC cells resistant to ABT-737 or ABT-263. We also demonstrate that MUC1-C is necessary for the resistance-associated increases in MCL-1 levels. Significantly, combining GO-203 with ABT-737 is synergistic in inhibiting survival of parental and drug resistant TNBC cells. These findings indicate that targeting MUC1-C is a potential strategy for reversing MCL-1-mediated resistance in TNBC. PMID:27217294

  12. HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

    PubMed Central

    Lecoeur, H; Borgne-Sanchez, A; Chaloin, O; El-Khoury, R; Brabant, M; Langonné, A; Porceddu, M; Brière, J-J; Buron, N; Rebouillat, D; Péchoux, C; Deniaud, A; Brenner, C; Briand, J-P; Muller, S; Rustin, P; Jacotot, E

    2012-01-01

    The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor. PMID:22419111

  13. Human self protein CD8+ T cell epitopes are both positively and negatively selected

    PubMed Central

    almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-01-01

    The cellular immune system recognizes self epitopes in the context of MHC-I molecules. The immunological general view presumes that these self epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self proteins types. Proteins presenting many epitopes include for example AIRE upregulated Tissue specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less “useful” for the immune system, such as low expression level proteins, are under presented. We combine our epitope estimate with SNP measures to show that this selection can be directly observed through the fraction of non-synonymous SNPs (replacement fraction), which is significantly higher inside epitopes than outside PMID:19291702

  14. Human self-protein CD8+ T-cell epitopes are both positively and negatively selected.

    PubMed

    Almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-04-01

    The cellular immune system recognizes self-epitopes in the context of MHC-I molecules. The immunological general view presumes that these self-epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self-protein types. Proteins presenting many epitopes include, for example, autoimmune regulator (AIRE) upregulated tissue-specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less "useful" for the immune system, such as low expression level proteins, are under-presented. We combine our epitope estimate with single nucleotide polymorphism (SNP) measures to show that this selection can be directly observed through the fraction of non-synonymous SNP (replacement fraction), which is significantly higher inside epitopes than outside. PMID:19291702

  15. Ras protein/cAMP-dependent protein kinase signaling is negatively regulated by a deubiquitinating enzyme, Ubp3, in yeast.

    PubMed

    Li, Yang; Wang, Yuqi

    2013-04-19

    Ras proteins and cAMP-dependent protein kinase (protein kinase A, PKA) are important components of a nutrient signaling pathway that mediates cellular responses to glucose in yeast. The molecular mechanisms that regulate Ras/PKA-mediated signaling remain to be fully understood. Here, we provide evidence that Ras/PKA signaling is negatively regulated by a deubiquitinating enzyme, Ubp3. Disrupting the activity of Ubp3 leads to hyperactivation of PKA, as evidenced by much enhanced phosphorylation of PKA substrates, decreased accumulation of glycogen, larger cell size, and increased sensitivity to heat shock. Levels of intracellular cAMP and the active forms of Ras proteins are also elevated in the ubp3Δ mutant. Consistent with a possibility that the increased cAMP is responsible for the abnormal signaling behavior of the ubp3Δ mutant, overexpressing PDE2, which encodes a phosphodiesterase that hydrolyzes cAMP, significantly relieves the cell size increase and heat shock sensitivity of the mutant. Further analysis reveals that Ubp3 interacts with a Ras GTPase-accelerating protein, Ira2, and regulates its level of ubiquitination. Together, our data indicate that Ubp3 is a new regulator of the Ras/PKA signaling pathway and suggest that Ubp3 regulates this pathway by controlling the ubiquitination of Ras GTPase-accelerating protein Ira2. PMID:23476013

  16. The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

    PubMed Central

    Govindarajan, Sutharsan; Elisha, Yair; Nevo-Dinur, Keren; Amster-Choder, Orna

    2013-01-01

    ABSTRACT The bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near the Escherichia coli cell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shaped E. coli cells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein from Bacillus subtilis at strongly curved sites in both B. subtilis and E. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness. PMID:24129255

  17. The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing

    PubMed Central

    Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang

    2015-01-01

    Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5−/− mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)–Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. PMID:26056233

  18. The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing.

    PubMed

    Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang; Choi, Kang-Yell

    2015-06-29

    Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5(-/-) mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)-Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. PMID:26056233

  19. Heat shock protein 90 stabilizes nucleolin to increase mRNA stability in mitosis.

    PubMed

    Wang, Shao-An; Li, Hao-Yi; Hsu, Tsung-I; Chen, Shu-Hui; Wu, Chin-Jen; Chang, Wen-Chang; Hung, Jan-Jong

    2011-12-23

    Most studies on heat shock protein 90 (Hsp90) have focused on the involvement of Hsp90 in the interphase, whereas the role of this protein in the nucleus during mitosis remains largely unclear. In this study, we found that the level of the acetylated form of Hsp90 decreased dramatically during mitosis, which indicates more chaperone activity during mitosis. We thus probed proteins that interacted with Hsp90 by liquid chromatography/mass spectrometry (LC/MS) and found that nucleolin was one of those interacting proteins during mitosis. The nucleolin level decreased upon geldanamycin treatment, and Hsp90 maintained the cyclin-dependent kinase 1 (CDK1) activity to phosphorylate nucleolin at Thr-641/707. Mutation of Thr-641/707 resulted in the destabilization of nucleolin in mitosis. We globally screened the level of mitotic mRNAs and found that 229 mRNAs decreased during mitosis in the presence of geldanamycin. Furthermore, a bioinformatics tool and an RNA immunoprecipitation assay found that 16 mRNAs, including cadherin and Bcl-xl, were stabilized through the recruitment of nucleolin to the 3'-untranslated regions (3'-UTRs) of those genes. Overall, strong correlations exist between the up-regulation of Hsp90, nucleolin, and the mRNAs related to tumorigenesis of the lung. Our findings thus indicate that nucleolin stabilized by Hsp90 contributes to the lung tumorigenesis by increasing the level of many tumor-related mRNAs during mitosis. PMID:21998300

  20. Protein secretion systems and adhesins: the molecular armory of Gram-negative pathogens.

    PubMed

    Gerlach, Roman G; Hensel, Michael

    2007-10-01

    Protein secretion is a basic cellular function found in organisms of all kingdoms of life. Gram-negative bacteria have evolved a remarkable number of pathways for the transport of proteins across the cell envelope. The secretion systems fulfill general cellular functions but are also essential for pathogenic bacteria during the interaction with eukaryotic host cells. Secretion systems range from relatively simple structures such as type I secretion systems composed of three subunits that only secrete one substrate protein to complex machines such as type III and IV secretion systems composed of more than 20 subunits that can translocate large sets of effector proteins into eukaryotic target cells. In this review, the main structural and functional features of secretion systems are described. One subgroup of substrate proteins of secretion systems are protein adhesins. Despite the conserved function in binding to host cell ligands or to abiotic surfaces, the assembly of the various bacterial adhesins is highly divergent. Here we give an overview on the recent understanding of the assembly of fimbrial and non-fimbrial adhesins and the role of type I, III and V secretion systems and specialized branches of the general secretion pathway in their biogenesis. PMID:17482513

  1. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat.

    PubMed

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na(+) and superfluous accumulation of Na(+) in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na(+)/H(+) exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  2. Bone Morphogenetic Protein 2 Signaling Negatively Modulates Lymphatic Development in Vertebrate Embryos

    PubMed Central

    Dunworth, William P.; Cardona-Costa, Jose; Bozkulak, Esra Cagavi; Kim, Jun-Dae; Meadows, Stryder; Fischer, Johanna C.; Wang, Yeqi; Cleaver, Ondine; Qyang, Yibing; Ober, Elke A.; Jin, Suk-Won

    2014-01-01

    Rationale The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. Objective Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. Methods and Results BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell–derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. Conclusions Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development. PMID:24122719

  3. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    PubMed Central

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na+/H+ exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  4. High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis.

    PubMed

    Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong

    2016-06-10

    HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. PMID:27154224

  5. Binding constraints on the evolution of enzymes and signalling proteins: the important role of negative pleiotropy.

    PubMed

    Liberles, David A; Tisdell, Makayla D M; Grahnen, Johan A

    2011-07-01

    A number of biophysical and population-genetic processes influence amino acid substitution rates. It is commonly recognized that proteins must fold into a native structure with preference over an unfolded state, and must bind to functional interacting partners favourably to function properly. What is less clear is how important folding and binding specificity are to amino acid substitution rates. A hypothesis of the importance of binding specificity in constraining sequence and functional evolution is presented. Examples include an evolutionary simulation of a population of SH2 sequences evolved by threading through the structure and binding to a native ligand, as well as SH3 domain signalling in yeast and selection for specificity in enzymatic reactions. An example in vampire bats where negative pleiotropy appears to have been adaptive is presented. Finally, considerations of compartmentalization and macromolecular crowding on negative pleiotropy are discussed. PMID:21490020

  6. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport

    PubMed Central

    Held, Aaron; Sargeant, John; Thorsen, Kevin; Hay, Jesse C.

    2016-01-01

    Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport—peflin is a negative regulator of transport. PMID:27276012

  7. Identification and functional characterization of Bet protein as a negative regulator of BFV3026 replication.

    PubMed

    Bing, Tiejun; Wu, Kai; Cui, Xiaoxu; Shao, Peng; Zhang, Qicheng; Bai, Xiaobo; Tan, Juan; Qiao, Wentao

    2014-06-01

    Foamy virus (FV) establishes persistent infection in the host without causing apparent disease. Besides the transactivator Tas protein, another auxiliary protein--Bet--has been reported in prototype foamy virus, equine foamy virus, and feline foamy virus. Here, we found the putative bbet gene in clone C74 from a cDNA library of bovine foamy virus strain 3026 (BFV3026) by comparison of gene localization, composition, and splicing features with other known bet genes. Subsequently, BBet protein was detected in BFV3026-infected cells by Western blot and immunofluorescence analyses. Analysis of the BBet mutant infectious clone (pBS-BFVdelBBet) revealed that BBet could inhibit BFV3026 replication. Consistent with this result, overexpression of BBet in Cf2Th cells reduced BFV replication by approximately threefold. Furthermore, virus replication levels similarly were reduced by approximately threefold in pBS-BFV-transfected and BFV3026-infected Cf2Th cells stably expressing BBet compared with control cells. After three passages, BFV3026 replicated more slowly in BBet-expressing cells. This study implicates BBet as a negative regulator of BFV replication and provides a resource for future studies on the function of this protein in the virus lifecycle. PMID:24615636

  8. Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

    PubMed Central

    Watkins, Herschel M.; Simon, Anna J.; Sosnick, Tobin R.; Lipman, Everett A.; Hjelm, Rex P.; Plaxco, Kevin W.

    2015-01-01

    Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant. PMID:25964362

  9. Protein-translocating outer membrane porins of Gram-negative bacteria.

    PubMed

    Yen, Ming Ren; Peabody, Christopher R; Partovi, Salar M; Zhai, Yufeng; Tseng, Yi Hsiung; Saier, Milton H

    2002-05-01

    Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins. PMID:11988218

  10. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    NASA Astrophysics Data System (ADS)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  11. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase. PMID:25131196

  12. A yeast BH3-only protein mediates the mitochondrial pathway of apoptosis

    PubMed Central

    Büttner, Sabrina; Ruli, Doris; Vögtle, F-Nora; Galluzzi, Lorenzo; Moitzi, Barbara; Eisenberg, Tobias; Kepp, Oliver; Habernig, Lukas; Carmona-Gutierrez, Didac; Rockenfeller, Patrick; Laun, Peter; Breitenbach, Michael; Khoury, Chamel; Fröhlich, Kai-Uwe; Rechberger, Gerald; Meisinger, Chris; Kroemer, Guido; Madeo, Frank

    2011-01-01

    Mitochondrial outer membrane permeabilization is a watershed event in the process of apoptosis, which is tightly regulated by a series of pro- and anti-apoptotic proteins belonging to the BCL-2 family, each characteristically possessing a BCL-2 homology domain 3 (BH3). Here, we identify a yeast protein (Ybh3p) that interacts with BCL-XL and harbours a functional BH3 domain. Upon lethal insult, Ybh3p translocates to mitochondria and triggers BH3 domain-dependent apoptosis. Ybh3p induces cell death and disruption of the mitochondrial transmembrane potential via the mitochondrial phosphate carrier Mir1p. Deletion of Mir1p and depletion of its human orthologue (SLC25A3/PHC) abolish stress-induced mitochondrial targeting of Ybh3p in yeast and that of BAX in human cells, respectively. Yeast cells lacking YBH3 display prolonged chronological and replicative lifespans and resistance to apoptosis induction. Thus, the yeast genome encodes a functional BH3 domain that induces cell death through phylogenetically conserved mechanisms. PMID:21673659

  13. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function

    PubMed Central

    Zhang, Yong; Zhao, Zhiyun; Ke, Bilun; Wan, Lin; Wang, Hui; Ye, Jianping

    2016-01-01

    It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function. PMID:26930489

  14. A Hypersweet Protein: Removal of The Specific Negative Charge at Asp21 Enhances Thaumatin Sweetness

    PubMed Central

    Masuda, Tetsuya; Ohta, Keisuke; Ojiro, Naoko; Murata, Kazuki; Mikami, Bunzo; Tani, Fumito; Temussi, Piero Andrea; Kitabatake, Naofumi

    2016-01-01

    Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain monellin, one of the two sweetest proteins known so far. The complex model between the T1R2-T1R3 sweet receptor and thaumatin, derived from tethered docking in the framework of the wedge model, confirmed that each of the positively charged residues critical for sweetness is close to a receptor residue of opposite charge to yield optimal electrostatic interaction. Furthermore, the distance between D21 and its possible counterpart D433 (located on the T1R2 protomer of the receptor) is safely large to avoid electrostatic repulsion but, at the same time, amenable to a closer approach if D21 is mutated into the corresponding asparagine. These findings clearly confirm the importance of electrostatic potentials in the interaction of thaumatin with the sweet receptor. PMID:26837600

  15. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila.

    PubMed Central

    Howard, S P; Buckley, J T

    1985-01-01

    The synthesis and export of aerolysin, an extracellular protein toxin released by the gram-negative bacterium Aeromonas hydrophila, was studied by pulse-labeling with [35S]methionine. The toxin was synthesized as a higher-molecular-weight precursor. This was processed cotranslationally, resulting in the appearance within the cell of the mature protein, which was then exported to the supernatant. Precursor aerolysin accumulated in cells incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone, a substance which also inhibited the export of mature aerolysin from the cell. The entrapped mature toxin could not be shocked from the cells, although it could be digested by protease applied to shocked cells. The toxin was processed and translocated across the inner membrane of pleiotropic export mutants and accumulated in the periplasm. The results indicate that more than one step is required for the export of the protein and that aerolysin does not cross the inner and outer membranes simultaneously. Images PMID:3972770

  16. Constitutive Negative Regulation of R Proteins in Arabidopsis also via Autophagy Related Pathway?

    PubMed Central

    Pečenková, Tamara; Sabol, Peter; Kulich, Ivan; Ortmannová, Jitka; Žárský, Viktor

    2016-01-01

    Even though resistance (R) genes are among the most studied components of the plant immunity, there remain still a lot of aspects to be explained about the regulation of their function. Many gain-of-function mutants of R genes and loss-of-function of their regulators often demonstrate up-regulated defense responses in combination with dwarf stature and/or spontaneous leaf lesions formation. For most of these mutants, phenotypes are a consequence of an ectopic activation of R genes. Based on the compilation and comparison of published results in this field, we have concluded that the constitutively activated defense phenotypes recurrently arise by disruption of tight, constitutive and multilevel negative control of some of R proteins that might involve also their targeting to the autophagy pathway. This mode of R protein regulation is supported also by protein–protein interactions listed in available databases, as well as in silico search for autophagy machinery interacting motifs. The suggested model could resolve some explanatory discrepancies found in the studies of the immunity responses of autophagy mutants. PMID:26973696

  17. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    PubMed Central

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  18. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell.

    PubMed

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  19. The negatively charged regions of lactoferrin binding protein B, an adaptation against anti-microbial peptides.

    PubMed

    Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B

    2014-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein's C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. PMID:24465982

  20. Direct stimulation of transcription by negative cofactor 2 (NC2) through TATA-binding protein (TBP)

    PubMed Central

    Cang, Yong; Prelich, Gregory

    2002-01-01

    Negative cofactor 2 (NC2) is an evolutionarily conserved transcriptional regulator that was originally identified as an inhibitor of basal transcription. Its inhibitory mechanism has been extensively characterized; NC2 binds to the TATA-binding protein (TBP), blocking the recruitment of TFIIA and TFIIB, and thereby inhibiting preinitiation complex assembly. NC2 is also required for expression of many yeast genes in vivo and stimulates TATA-less transcription in a Drosophila in vitro transcription system, but the mechanism responsible for the NC2-mediated stimulation of transcription is not understood. Here we establish that yeast NC2 can directly stimulate activated transcription from TATA-driven promoters both in vivo and in vitro, and moreover that this positive role requires the same surface of TBP that mediates the NC2 repression activity. On the basis of these results, we propose a model to explain how NC2 can mediate both repression and activation through the same surface of TBP. PMID:12237409

  1. Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase

    PubMed Central

    Tong, Amy; Lynn, Grace; Ngo, Vy; Wong, Daniel; Moseley, Sarah L.; Ewbank, Jonathan J.; Goncharov, Alexandr; Wu, Yi-Chun; Pujol, Nathalie; Chisholm, Andrew D.

    2009-01-01

    Wounding of epidermal layers triggers multiple coordinated responses to damage. We show here that the Caenorhabditis elegans ortholog of the tumor suppressor death-associated protein kinase, dapk-1, acts as a previously undescribed negative regulator of barrier repair and innate immune responses to wounding. Loss of DAPK-1 function results in constitutive formation of scar-like structures in the cuticle, and up-regulation of innate immune responses to damage. Overexpression of DAPK-1 represses innate immune responses to needle wounding. Up-regulation of innate immune responses in dapk-1 requires the TIR-1/p38 signal transduction pathway; loss of function in this pathway synergizes with dapk-1 to drastically reduce adult lifespan. Our results reveal a previously undescribed function for the DAPK tumor suppressor family in regulation of epithelial damage responses. PMID:19164535

  2. The β-barrel membrane protein insertase machinery from Gram-negative bacteria

    PubMed Central

    Noinaj, Nicholas; Rollauer, Sarah E.; Buchanan, Susan K.

    2015-01-01

    The outer membranes (OM) of Gram-negative bacteria contain a host of β-barrel outer membrane proteins (OMPs) which serve many functions for cell survival and virulence. The biogenesis of these OMPs is mediated by the β-barrel assembly machinery (BAM) complex which is composed of five components including the essential core component called BamA that mediates the insertase function within the OM. The crystal structure of BamA has recently been reported from three different species, including a full-length structure from Neisseria gonorrhoeae. Mutagenesis and functional studies identified several conformational changes within BamA that are required for function, providing a significant advancement towards unraveling exactly how BamA and the BAM complex are able to fold and insert new OMPs in the OM. PMID:25796031

  3. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.

    PubMed

    Couto, Daniel; Niebergall, Roda; Liang, Xiangxiu; Bücherl, Christoph A; Sklenar, Jan; Macho, Alberto P; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Maclean, Dan; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min; Zipfel, Cyril

    2016-08-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  4. Positive Charge of “Sticky” Peptides and Proteins Impedes Release From Negatively Charged PLGA Matrices

    PubMed Central

    Balmert, Stephen C.; Zmolek, Andrew C.; Glowacki, Andrew J.; Knab, Timothy D.; Rothstein, Sam N.; Wokpetah, Joseph M.; Fedorchak, Morgan V.; Little, Steven R.

    2015-01-01

    The influence of electrostatic interactions and/or acylation on release of charged (“sticky”) agents from biodegradable polymer matrices was systematically characterized. We hypothesized that release of peptides with positive charge would be hindered from negatively charged poly(lactic-co-glycolic acid) (PLGA) microparticles. Thus, we investigated release of peptides with different degrees of positive charge from several PLGA microparticle formulations, with different molecular weights and/or end groups (acid- or ester-terminated). Indeed, release studies revealed distinct inverse correlations between the amount of positive charge on peptides and their release rates from each PLGA microparticle formulation. Furthermore, we examined the case of peptides with net charge that changes from negative to positive within the pH range observed in degrading microparticles. These charge changing peptides displayed counterintuitive release kinetics, initially releasing faster from slower degrading (less acidic) microparticles, and releasing slower from the faster degrading (more acidic) microparticles. Importantly, trends between agent charge and release rates for model peptides also translated to larger, therapeutically relevant proteins and oligonucleotides. The results of these studies may improve future design of controlled release systems for numerous therapeutic biomolecules exhibiting positive charge, ultimately reducing time-consuming and costly trial and error iterations of such formulations. PMID:26085928

  5. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1

    PubMed Central

    Liang, Xiangxiu; Bücherl, Christoph A.; Sklenar, Jan; Macho, Alberto P.; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min

    2016-01-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  6. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    PubMed

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  7. MDM1 is a microtubule-binding protein that negatively regulates centriole duplication

    PubMed Central

    Van de Mark, Daniel; Kong, Dong; Loncarek, Jadranka; Stearns, Tim

    2015-01-01

    Mouse double-minute 1 (Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding. PMID:26337392

  8. A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

    PubMed

    Lorenzato, Annalisa; Olivero, Martina; Perro, Mario; Brière, Jean Jacques; Rustin, Pierre; Di Renzo, Maria Flavia

    2008-02-15

    The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation. PMID:17960613

  9. Characterization of a highly negative and labile binding protein induced in Euglena gracilis by cadmium

    SciTech Connect

    Gingrich, D.J.; Weber, D.N.; Shaw, C.F.; Garvey, J.S.; Petering, D.H.

    1986-03-01

    The physiochemical properties and physiological significance of the cadmium-binding protein (CdBP) of the algae Euglena gracilis have been studied. Following in vivo exposure of cells to 0.4 or 1.3 ..mu..g/mL of Cd/sup 2 +/, all the cytosolic Cd is bound to high molecular weight species. At 4.7 ..mu..g/mL, appreciable CdBP has formed in cells grown under illumination or in the dark. The large pool of very low molecular weight zinc species previously reported is increased when cells are exposed to high cadmium levels. Two distinct species, BP-1 and BP-2 are resolved by ion-exchange chromatography on DEAE-Sephadex. Unusually high conductivities are required to displace them, indicating that they are very negatively charged proteins at pH 8.6. The pH for half-titration of bound Cd/sup 2 +/ is between 5 and 6. Neither form of the CdBP cross-reacts with antibodies to rat liver metallothionein (MT) antibodies. The structural, chemical, and functional differences between the Euglena CdBPs and mammalian MTs are discussed. When cells are exposed to high levels of Cu, a CuBP is induced, and the very low molecular weight zinc band is depleted.

  10. The Negatively Charged Regions of Lactoferrin Binding Protein B, an Adaptation against Anti-Microbial Peptides

    PubMed Central

    Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B.

    2014-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. PMID:24465982

  11. Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.

    PubMed

    Wang, Fang; Weng, Hongbo; Quon, Michael J; Yu, Jingwen; Wang, Jian-Ying; Hueber, Anne-Odile; Yang, Peixin

    2015-11-15

    Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress. PMID:26419589

  12. Genome-wide inference of protein interaction sites: lessons from the yeast high-quality negative protein–protein interaction dataset

    PubMed Central

    Guo, Jie; Wu, Xiaomei; Zhang, Da-Yong; Lin, Kui

    2008-01-01

    High-throughput studies of protein interactions may have produced, experimentally and computationally, the most comprehensive protein–protein interaction datasets in the completely sequenced genomes. It provides us an opportunity on a proteome scale, to discover the underlying protein interaction patterns. Here, we propose an approach to discovering motif pairs at interaction sites (often 3–8 residues) that are essential for understanding protein functions and helpful for the rational design of protein engineering and folding experiments. A gold standard positive (interacting) dataset and a gold standard negative (non-interacting) dataset were mined to infer the interacting motif pairs that are significantly overrepresented in the positive dataset compared to the negative dataset. Four negative datasets assembled by different strategies were evaluated and the one with the best performance was used as the gold standard negatives for further analysis. Meanwhile, to assess the efficiency of our method in detecting potential interacting motif pairs, other approaches developed previously were compared, and we found that our method achieved the highest prediction accuracy. In addition, many uncharacterized motif pairs of interest were found to be functional with experimental evidence in other species. This investigation demonstrates the important effects of a high-quality negative dataset on the performance of such statistical inference. PMID:18281313

  13. BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

    PubMed Central

    Hafeez, Samra; Urooj, Mahwish; Saleem, Shamiala; Gillani, Zeeshan; Shaheen, Sumaira; Qazi, Mahmood Husain; Naseer, Muhammad Imran; Iqbal, Zafar; Ansari, Shakeel Ahmed; Haque, Absarul; Asif, Muhammad; Mir, Manzoor Ahmad; Ali, Ashraf; Pushparaj, Peter Natesan; Jamal, Mohammad Sarwar; Rasool, Mahmood

    2016-01-01

    This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent. PMID:26745145

  14. Adaptor protein LNK is a negative regulator of brain neural stem cell proliferation after stroke.

    PubMed

    Ahlenius, Henrik; Devaraju, Karthikeyan; Monni, Emanuela; Oki, Koichi; Wattananit, Somsak; Darsalia, Vladimer; Iosif, Robert E; Torper, Olof; Wood, James C; Braun, Sebastian; Jagemann, Lucas; Nuber, Ulrike A; Englund, Elisabet; Jacobsen, Sten-Eirik W; Lindvall, Olle; Kokaia, Zaal

    2012-04-11

    Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain. PMID:22496561

  15. Negative regulation of parathyroid hormone-related protein expression by steroid hormones

    SciTech Connect

    Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko; Chikamori, Minoru; Iizuka, Masayoshi; Okazaki, Tomoki

    2011-04-15

    Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.

  16. Nigrostriatal alterations in bone morphogenetic protein receptor II dominant negative mice

    PubMed Central

    Chou, J.; Harvey, B. K.; Ebendal, T.; Hoffer, B.; Wang, Y.

    2008-01-01

    Summary Background We previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson’s disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. Methods Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. Results BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. Conclusions Endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. PMID:18642641

  17. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins.

    PubMed

    Peng, Jamy C; Valouev, Anton; Liu, Na; Lin, Haifan

    2016-03-01

    The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis. PMID:26780607

  18. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb Group proteins

    PubMed Central

    Peng, Jamy C.; Valouev, Anton; Liu, Na; Lin, Haifan

    2015-01-01

    The Drosophila Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi cooperates with Polycomb Group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin co-immunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), lysine-27-tri-methylated histone 3 (H3K27m3), and RNA polymerase II in wild-type and piwi mutant ovaries reveals that Piwi binds a conserved DNA motif at ~72 genomic sites, and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 tri-methylation. Moreover, Piwi influences RNA Polymerase II activities in Drosophila ovaries likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influences transcription during oogenesis. PMID:26780607

  19. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons

    SciTech Connect

    Helmann, J.D.; Walsh, C.T. ); Wang, Ying; Mahler, I. )

    1989-01-01

    The authors report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator region in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.

  20. Protein functional properties prediction in sparsely-label PPI networks through regularized non-negative matrix factorization

    PubMed Central

    2015-01-01

    Background Predicting functional properties of proteins in protein-protein interaction (PPI) networks presents a challenging problem and has important implication in computational biology. Collective classification (CC) that utilizes both attribute features and relational information to jointly classify related proteins in PPI networks has been shown to be a powerful computational method for this problem setting. Enabling CC usually increases accuracy when given a fully-labeled PPI network with a large amount of labeled data. However, such labels can be difficult to obtain in many real-world PPI networks in which there are usually only a limited number of labeled proteins and there are a large amount of unlabeled proteins. In this case, most of the unlabeled proteins may not connected to the labeled ones, the supervision knowledge cannot be obtained effectively from local network connections. As a consequence, learning a CC model in sparsely-labeled PPI networks can lead to poor performance. Results We investigate a latent graph approach for finding an integration latent graph by exploiting various latent linkages and judiciously integrate the investigated linkages to link (separate) the proteins with similar (different) functions. We develop a regularized non-negative matrix factorization (RNMF) algorithm for CC to make protein functional properties prediction by utilizing various data sources that are available in this problem setting, including attribute features, latent graph, and unlabeled data information. In RNMF, a label matrix factorization term and a network regularization term are incorporated into the non-negative matrix factorization (NMF) objective function to seek a matrix factorization that respects the network structure and label information for classification prediction. Conclusion Experimental results on KDD Cup tasks predicting the localization and functions of proteins to yeast genes demonstrate the effectiveness of the proposed RNMF method for

  1. MetaLocGramN: A meta-predictor of protein subcellular localization for Gram-negative bacteria.

    PubMed

    Magnus, Marcin; Pawlowski, Marcin; Bujnicki, Janusz M

    2012-12-01

    Subcellular localization is a key functional characteristic of proteins. It is determined by signals encoded in the protein sequence. The experimental determination of subcellular localization is laborious. Thus, a number of computational methods have been developed to predict the protein location from sequence. However predictions made by different methods often disagree with each other and it is not always clear which algorithm performs best for the given cellular compartment. We benchmarked primary subcellular localization predictors for proteins from Gram-negative bacteria, PSORTb3, PSLpred, CELLO, and SOSUI-GramN, on a common dataset that included 1056 proteins. We found that PSORTb3 performs best on the average, but is outperformed by other methods in predictions of extracellular proteins. This motivated us to develop a meta-predictor, which combines the primary methods by using the logistic regression models, to take advantage of their combined strengths, and to eliminate their individual weaknesses. MetaLocGramN runs the primary methods, and based on their output classifies protein sequences into one of five major localizations of the Gram-negative bacterial cell: cytoplasm, plasma membrane, periplasm, outer membrane, and extracellular space. MetaLocGramN achieves the average Matthews correlation coefficient of 0.806, i.e. 12% better than the best individual primary method. MetaLocGramN is a meta-predictor specialized in predicting subcellular localization for proteins from Gram-negative bacteria. According to our benchmark, it performs better than all other tools run independently. MetaLocGramN is a web and SOAP server available for free use by all academic users at the URL http://iimcb.genesilico.pl/MetaLocGramN. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction. PMID:22705560

  2. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.

    PubMed Central

    Zhang, Y; Rollins, B J

    1995-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs. PMID:7651403

  3. The DNA binding domain of p53 is sufficient to trigger a potent apoptotic response at the mitochondria.

    PubMed

    Matissek, Karina J; Mossalam, Mohanad; Okal, Abood; Lim, Carol S

    2013-10-01

    The tumor suppressor p53 is one of the most studied proteins in human cancer.1-3 While nuclear p53 has been utilized for cancer gene therapy, mitochondrial targeting of p53 has not been fully exploited to date.4,5 In response to cellular stress, p53 translocates to the mitochondria and directly interacts with Bcl-2 family proteins including antiapoptotic Bcl-XL and Bcl-2 and proapoptotic Bak and Bax.6 Antiapoptotic Bcl-XL forms inhibitory complexes with proapoptotic Bak and Bax preventing their homo-oligomerization.7 Upon translocation to the mitochondria, p53 binds to Bcl-XL, releases Bak and Bax from the inhibitory complex and enhances their homo-oligomerization.8 Bak and Bax homotetramer formation disrupts the mitochondrial outer membrane, releases antiapoptotic factors such as cytochrome c and triggers a rapid apoptotic response mediated by caspase induction.9 It is still unclear if the MDM2 binding domain (MBD), the proline-rich domain (PRD) and/or DNA binding domain (DBD) of p53 are the domains responsible for interaction with Bcl-XL.10-17 The purpose of this work is to determine if a smaller functional domain of p53 is capable of inducing apoptosis similarly to full length p53. To explore this question, different domains of p53 (MBD, PRD, DBD) were fused to the mitochondrial targeting signal (MTS) from Bcl-XL to ensure Bcl-XL specific targeting.18 The designed constructs were tested for apoptotic activity (TUNEL, Annexin-V, and 7-AAD) in 3 different breast cancer cell lines (T47D, MCF-7, MDA-MB-231), in a cervical cancer cell line (HeLa) and in non-small cell lung adenocarcinoma cells H1373. Our results indicate that DBD-XL (p53 DBD fused to the Bcl-XL MTS) reproduces (in T47D cells) or demonstrates increased apoptotic activity (in MCF-7, MDA-MB-231, and HeLa cells) compared to p53-XL (full length p53 fused to Bcl-XL MTS). Additionally, mitochondrial dependent apoptosis assays (TMRE, caspase-9), co-IP and overexpression of Bcl-XL in T47D cells suggest that

  4. Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts

    PubMed Central

    Misra, Rajeev

    2012-01-01

    In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts.

  5. Systems and methods for the secretion of recombinant proteins in gram negative bacteria

    DOEpatents

    Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

    2016-08-09

    Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

  6. Rhomboid domain-containing protein 3 is a negative regulator of TLR3-triggered natural killer cell activation.

    PubMed

    Liu, Juan; Liu, Shuxun; Xia, Meng; Xu, Sheng; Wang, Chunmei; Bao, Yan; Jiang, Minghong; Wu, Yue; Xu, Tian; Cao, Xuetao

    2013-05-01

    Rhomboid domain-containing protein 3 (Rhbdd3), which belongs to a family of proteins with rhomboid domain, is widely expressed in immune cells; however, the roles of the Rhbdd members, including Rhbdd3, in immunity remain unknown. Natural killer (NK) cells are critical for host immune defense and also can mediate inflammatory diseases such as hepatitis. Although much is known about how NK cells are activated, the detailed mechanisms for negative regulation of NK cell activation remain to be fully understood. Using Rhbdd3-deficient mice, we reveal that Rhbdd3, selectively up-regulated in NK cells upon Toll-like receptor 3 (TLR3) stimulation, negatively regulates TLR3-mediated NK cell activation in a feedback manner. Rhbdd3 inhibits TLR3-triggered IFN-γ and granzyme B expression of NK cells in cell-cell contact dependence of accessory cells such as dendritic cells and Kupffer cells. Rhbdd3 interacts with DNAX activation protein of 12 kDa and promotes its degradation, inhibiting MAPK activation in TLR3-triggered NK cells. Furthermore, Rhbdd3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in liver and disrupting NK cell-Kupffer cell interaction. Therefore, Rhbdd3 is a feedback inhibitor of TLR3-triggered NK cell activation. Our study outlines a mechanism for the negative regulation of NK cell activation and also provides clues for the function of the rhomboid proteins in immunity. PMID:23610400

  7. Molecular characterization of SIG1, a Saccharomyces cerevisiae gene involved in negative regulation of G-protein-mediated signal transduction.

    PubMed Central

    Leberer, E; Dignard, D; Harcus, D; Whiteway, M; Thomas, D Y

    1994-01-01

    Two recessive mutations in the Saccharomyces cerevisiae SIG1 (suppressor of inhibitory G-protein) gene have been identified by their ability to suppress the signalling defect of dominant-negative variants of the mating response G-protein beta-subunit. The mutations and deletion of SIG1 enhance the sensitivity of the cells to pheromone and stimulate the basal transcription of a mating specific gene, FUS1, suggesting that Sig1p plays a negatively regulatory role in G beta gamma-mediated signal transduction. An additional function of Sig1p in vegetatively growing cells is suggested by the finding that the mutations and deletion of SIG1 cause temperature-sensitive growth defects. The SIG1 gene encodes a protein with a molecular weight of 65 kDa that contains at the amino-terminus two zinc finger-like sequence motifs. Epistasis experiments localize the action of Sig1p within the pheromone signalling pathway at a position at or shortly after the G-protein. We propose that Sig1p represents a novel negative regulator of G beta gamma-mediated signal transduction. Images PMID:8039500

  8. Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

    PubMed

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001). PMID:27335148

  9. Identification of the innate human immune response to surface-exposed proteins of coagulase-negative staphylococci.

    PubMed Central

    Plaunt, M R; Patrick, C C

    1991-01-01

    The presumed host defense against coagulase-negative staphylococci (ConS), recognized pathogens in hosts with compromised immunity or indwelling medical devices, is opsonophagocytosis. Targets for opsonization remain unclear. Using radiolabeling techniques, we identified the surface-exposed proteins of ConS and determined the innate humoral immune responses to them among healthy adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface proteins extrinsically labeled with 125I demonstrated 20 to 30 proteins with molecular weights of 15,000 to greater than 130,000. Five to ten of these proteins were immunogenic and recognized by normal human sera, including predominant 18-, 41-, 48-, and 51-kDa proteins. We also evaluated the humoral response of cancer patients with ConS bacteremia. Patients' sera obtained before bacteremic episodes demonstrated a pattern of reactivity similar to that of normal human sera. When patients' sera obtained after bacteremic episodes were used to determine whether an expanded immune response followed infection, only one of seven showed reactivity with more proteins than seen with the innate response. Western blot (immunoblot) analysis and whole-cell enzyme-linked immunosorbent assays were also evaluated. This study identifies (i) the surface-exposed proteins available for host interaction, (ii) the innate human antibody response to these proteins, and (iii) the immune response of cancer patients with ConS bacteremia. Images PMID:2056051

  10. The nuclear localization of SOCS6 requires the N-terminal region and negatively regulates Stat3 protein levels

    SciTech Connect

    Hwang, Mi-Na; Min, Chan-Hee; Kim, Hyung Sik; Lee, Ho; Yoon, Kyong-Ah; Park, Sung Yong; Lee, Eun Sook; Yoon, Sungpil . E-mail: yoons@ncc.re.kr

    2007-08-24

    We determined that endogenous- and overexpressed- SOCS6 was localized in both the nucleus and cytoplasm. The localization of SOCS6 depended on amino acids 1-210 in the N-terminal region of the protein, which contains an unidentified domain. GFP-tagged SOCS6 or the N-terminal region, was exclusively localized and widely distributed throughout the entire nucleus, whereas the C-terminal region displayed a nuclear omission pattern. We also demonstrated that the SOCS6 protein could decrease the levels of the Stat3 protein in the nucleus, and that its negative regulation of the Stat3 protein level was dependent on its C-terminal region. These observations suggest that SOCS6 is composed of at least two functional domains required for its biological role in localizing and degrading Stat3 in the nucleus.

  11. A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy

    PubMed Central

    DeMaster, Laura K.; Liu, Xiaohe; VanBelzen, D. Jake; Trinité, Benjamin; Zheng, Lingjie; Agosto, Luis M.; Migueles, Stephen A.; Connors, Mark; Sambucetti, Lidia; Levy, David N.; Pasternak, Alexander O.

    2015-01-01

    ABSTRACT A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4− cells. In this study, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag+ CD4− cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs. PMID:26537682

  12. Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

    PubMed Central

    Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

    2012-01-01

    Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

  13. Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells.

    PubMed

    Gao, Jie; Ma, Yue; Fu, Hua-Lin; Luo, Qian; Wang, Zhen; Xiao, Yu-Huan; Yang, Hao; Cui, Da-Xiang; Jin, Wei-Lin

    2016-05-01

    The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma. PMID:27113584

  14. Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein.

    PubMed

    Verma, Sandhya; Bednar, Valerie; Blount, Andrew; Hogue, Brenda G

    2006-05-01

    The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D441-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly. PMID:16611893

  15. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  16. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  17. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  18. Heat Modifiability of Outer Membrane Proteins from Gram-Negative Bacteria

    PubMed Central

    Noinaj, Nicholas; Kuszak, Adam J.; Buchanan, Susan K.

    2016-01-01

    Summary β-barrel membrane proteins are somewhat unique in that their folding states can be monitored using semi-native SDS-PAGE methods to determine if they are folded properly or not. This property, which is commonly referred to as heat modifiability, has been used for many years on both purified protein and on whole cells to monitor folded states of proteins of interest. Additionally, heat modifiability assays have proven indispensable in studying the BAM complex and its role in folding and inserting β-barrel membrane proteins into the outer membrane. Here, we describe the protocol our lab uses for performing the heat modifiability assay in our studies on outer membrane proteins. PMID:26427675

  19. Heat Modifiability of Outer Membrane Proteins from Gram-Negative Bacteria.

    PubMed

    Noinaj, Nicholas; Kuszak, Adam J; Buchanan, Susan K

    2015-01-01

    β-barrel membrane proteins are somewhat unique in that their folding states can be monitored using semi-native SDS-PAGE methods to determine if they are folded properly or not. This property, which is commonly referred to as heat modifiability, has been used for many years on both purified protein and on whole cells to monitor folded states of proteins of interest. Additionally, heat modifiability assays have proven indispensable in studying the BAM complex and its role in folding and inserting β-barrel membrane proteins into the outer membrane. Here, we describe the protocol our lab uses for performing the heat modifiability assay in our studies on outer membrane proteins. PMID:26427675

  20. Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein.

    PubMed

    Weise, Christoph F; Login, Frédéric H; Ho, Oanh; Gröbner, Gerhard; Wolf-Watz, Hans; Wolf-Watz, Magnus

    2014-10-21

    The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia. PMID:25418176

  1. The role of negative selection in protein evolution revealed through the energetics of the native state ensemble.

    PubMed

    Hoffmann, Jordan; Wrabl, James O; Hilser, Vincent J

    2016-04-01

    Knowing the determinants of conformational specificity is essential for understanding protein structure, stability, and fold evolution. To address this issue, a novel statistical measure of energetic compatibility between sequence and structure was developed using an experimentally validated model of the energetics of the native state ensemble. This approach successfully matched sequences from a diverse subset of the human proteome to their respective folds. Unexpectedly, significant energetic compatibility between ostensibly unrelated sequences and structures was also observed. Interrogation of these matches revealed a general framework for understanding the origins of conformational specificity within a proteome: specificity is a complex function of both the ability of a sequence to adopt folds other than the native, and ability of a fold to accommodate sequences other than the native. The regional variation in energetic compatibility indicates that the compatibility is dominated by incompatibility of sequence for alternative fold segments, suggesting that evolution of protein sequences has involved substantial negative selection, with certain segments serving as "gatekeepers" that presumably prevent alternative structures. Beyond these global trends, a size dependence exists in the degree to which the energetic compatibility is determined from negative selection, with smaller proteins displaying more negative selection. This partially explains how short sequences can adopt unique folds, despite the higher probability in shorter proteins for small numbers of mutations to increase compatibility with other folds. In providing evolutionary ground rules for the thermodynamic relationship between sequence and fold, this framework imparts valuable insight for rational design of unique folds or fold switches. Proteins 2016; 84:435-447. © 2016 Wiley Periodicals, Inc. PMID:26800099

  2. Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation

    PubMed Central

    Lin, Jing-Yi; Li, Mei-Ling; Shih, Shin-Ru

    2009-01-01

    An internal ribosomal entry site (IRES) that directs the initiation of viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the internal ribosomal entry site. Biotinylated RNA-affinity chromatography and proteomic approaches were employed to identify far upstream element (FUSE) binding protein 2 (FBP2) as an ITAF for EV71. The interactions of FBP2 with EV71 IRES were confirmed by competition assay and by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in the nucleus in mock-infected cells. The synthesis of viral proteins increased in FBP2-knockdown cells that were infected by EV71. IRES activity in FBP2-knockdown cells exceeded that in the negative control (NC) siRNA-treated cells. On the other hand, IRES activity decreased when FBP2 was over-expressed in the cells. Results of this study suggest that FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation. PMID:19010963

  3. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

    PubMed Central

    Rames, Matthew; Yu, Yadong; Ren, Gang

    2014-01-01

    Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and

  4. Regulation of OSU-03012 toxicity by ER stress proteins and ER stress inducing drugs

    PubMed Central

    Booth, Laurence; Roberts, Jane L.; Cruickshanks, Nichola; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and to determine the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knock down of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase 9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase 8 inhibitor c-FLIP-s, or knock down of death receptor CD95 or the death receptor – caspase 8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knock down of the autophagy regulatory proteins Beclin1 or ATG5 protected cells from OSU-03012 and of [OSU-03012 + PDE5 inhibitor] toxicity. Knock down of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor] –induced JNK activation and inhibition of JNK suppressed the elevated killing caused by IRE1 knock down. Knock down of CD95 blunted JNK activation. Collectively our data demonstrates that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in GBM cells. PMID:25103559

  5. The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy.

    PubMed

    Postis, Vincent; Rawson, Shaun; Mitchell, Jennifer K; Lee, Sarah C; Parslow, Rosemary A; Dafforn, Tim R; Baldwin, Stephen A; Muench, Stephen P

    2015-02-01

    Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function. PMID:25450810

  6. Interferonβ-1b Induces the Expression of RGS1 a Negative Regulator of G-Protein Signaling

    PubMed Central

    Tran, Tiffany; Paz, Pedro; Velichko, Sharlene; Cifrese, Jill; Belur, Praveen; Yamaguchi, Ken D.; Ku, Karin; Mirshahpanah, Parham; Reder, Anthony T.; Croze, Ed

    2010-01-01

    We present evidence of a link between interferonβ-1b (IFN-β) and G-protein signaling by demonstrating that IFN-β can induce the expression of the negative regulator of G-protein signaling 1 (RGS1). RGS1 reduces G-protein activation and immune cell migration by interacting with heterotrimeric G-proteins and enhancing their intrinsic GTPase activity. In this study, IFN-β treatment resulted in the induction of RGS1 in peripheral blood mononuclear cells (PBMCs), monocytes, T cells, and B cells. Induction of RGS1 by IFN-β was concentration dependent and observed at both the RNA and protein level. Other members of the RGS family were not induced by IFN-β, and induction of RGS1 required the activation of the IFN receptor. In addition, RGS1 induction was observed in PBMCs obtained from IFN-β-treated multiple sclerosis patients suggesting a possible, as yet unexplored, involvement of G-protein regulation in disease treatment. The upregulation of RGS1 by IFN-β has not been previously reported. PMID:21274427

  7. Negative effects of desiccation on the protein sorting and post-translational modification.

    PubMed

    Wang, Xiaoqin; He, Yikun

    2009-05-01

    Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress. In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition. PMID:19816114

  8. Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

    PubMed Central

    Draberova, Lubica; Bugajev, Viktor; Potuckova, Lucie; Halova, Ivana; Bambouskova, Monika; Polakovicova, Iva; Xavier, Ramnik J.; Seed, Brian

    2014-01-01

    The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. PMID:25246632

  9. The negative transcription factor E4BP4 is associated with circadian clock protein PERIOD2.

    PubMed

    Ohno, Tomoya; Onishi, Yoshiaki; Ishida, Norio

    2007-03-23

    The bZIP transcription factor E4BP4, is a mammalian homologue of vrille that functions as a key negative component of the circadian clock. We have shown that the E4BP4-binding site (B-site) is required in addition to a non-canonical E-box (E2 enhancer) for robust circadian Period2 (Per2) expression in the cell-autonomous clock. While the E2 enhancer and the B-site are closely situated, correlations between each component bound to the E2 enhancer and the B-site remain obscure. Here, we show that E4BP4 interacts with PER2, which represses transcriptional activity via the E-box enhancer. Interaction with PER2 required the carboxyl-terminal region that contains the repression domain of E4BP4. We also found that E4BP4 interacts with CRYPTOCHROME2 (CRY2), a key negative regulator in the mammalian circadian clock. These results suggest that E4BP4 is a component of the negative regulator complex of mammalian circadian clocks. PMID:17274955

  10. Pig α1-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein

    PubMed Central

    Heegaard, Peter M. H.; Miller, Ingrid; Sorensen, Nanna Skall; Soerensen, Karen Elisabeth; Skovgaard, Kerstin

    2013-01-01

    The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1–3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2–5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein. PMID:23844161

  11. Dynamin-2 is a novel NOS1β interacting protein and negative regulator in the collecting duct.

    PubMed

    Hyndman, Kelly A; Arguello, Alexandra M; Morsing, Sofia K H; Pollock, Jennifer S

    2016-04-01

    Nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) production in collecting ducts is critical for maintaining fluid-electrolyte balance. Rat collecting ducts express both the full-length NOS1α and its truncated variant NOS1β, while NOS1β predominates in mouse collecting ducts. We reported that dynamin-2 (DNM2), a protein involved in excising vesicles from the plasma membrane, and NOS1α form a protein-protein interaction that promotes NO production in rat collecting ducts. NOS1β was found to be highly expressed in human renal cortical/medullary samples; hence, we tested the hypothesis that DNM2 is a positive regulator of NOS1β-derived NO production. COS7 and mouse inner medullary collecting duct-3 (mIMCD3) cells were transfected with NOS1β and/or DNM2. Coimmunoprecipitation experiments show that NOS1β and DNM2 formed a protein-protein interaction. DNM2 overexpression decreased nitrite production (index of NO) in both COS7 and mIMCD-3 cells by 50-75%. mIMCD-3 cells treated with a panel of dynamin inhibitors or DNM2 siRNA displayed increased nitrite production. To elucidate the physiological significance of IMCD DNM2/NOS1β regulation in vivo, flox control and CDNOS1 knockout mice were placed on a high-salt diet, and freshly isolated IMCDs were treated acutely with a dynamin inhibitor. Dynamin inhibition increased nitrite production by IMCDs from flox mice. This response was blunted (but not abolished) in collecting duct-specific NOS1 knockout mice, suggesting that DNM2 also negatively regulates NOS3 in the mouse IMCD. We conclude that DNM2 is a novel negative regulator of NO production in mouse collecting ducts. We propose that DNM2 acts as a "break" to prevent excess or potentially toxic NO levels under high-salt conditions. PMID:26791826

  12. Negative feedback in the bone morphogenetic protein 4 (BMP4) synexpression group governs its dynamic signaling range and canalizes development

    PubMed Central

    Paulsen, Malte; Legewie, Stefan; Eils, Roland; Karaulanov, Emil; Niehrs, Christof

    2011-01-01

    What makes embryogenesis a robust and canalized process is an important question in developmental biology. A bone morphogenetic protein (BMP) morphogen gradient plays a key role in embryonic development, and we are beginning to understand how the self-regulating properties of its signaling circuitry ensure robust embryonic patterning. An unexplored question is why the BMP signaling circuit is organized as a modular synexpression group, with a prevalence of feedback inhibitors. Here, we provide evidence from direct experimentation and mathematical modeling that the synexpressed feedback inhibitors BAMBI, SMAD6, and SMAD7 (i) expand the dynamic BMP signaling range essential for proper embryonic patterning and (ii) reduce interindividual phenotypic and molecular variability in Xenopus embryos. Thereby, negative feedback linearizes signaling responses and confers robust patterning, thus promoting canalized development. The presence of negative feedback inhibitors in other growth factor synexpression groups suggests that these properties may constitute a general principle. PMID:21633009

  13. MecA Protein Acts as a Negative Regulator of Genetic Competence in Streptococcus mutans

    PubMed Central

    Tian, Xiao-Lin; Dong, Gaofeng; Liu, Tianlei; Gomez, Zubelda A.; Wahl, Astrid; Hols, Pascal

    2013-01-01

    Streptococcus mutans develops competence for genetic transformation through a complex network that receives inputs from at least two signaling peptides, competence-stimulating peptide (CSP) and sigX-inducing peptide (XIP). The key step of competence induction is the transcriptional activation of comX, which encodes an alternative sigma factor, SigX (σX), controlling the expression of late competence genes essential for DNA uptake and recombination. In this study, we provide evidence that MecA acts as a negative regulator in the posttranslational regulation of SigX in S. mutans. Using luxAB transcriptional reporter strains, we demonstrate that MecA represses the expression of late competence genes in S. mutans grown in a complex medium that is subpermissive for competence induction by CSP. The negative regulation of competence by MecA requires the presence of a functional SigX. Accordingly, inactivation of MecA results in a prolonged competence state of S. mutans under this condition. We have also found that the AAA+ protease ClpC displays a similar repressing effect on late competence genes, suggesting that both MecA and ClpC function coordinately to regulate competence in the same regulatory circuit in S. mutans. This suggestion is strongly supported by the results of bacterial two-hybrid assays, which demonstrate that MecA interacts with both SigX and ClpC, forming a ternary SigX-MecA-ClpC complex. Western blot analysis also confirms that inactivation of MecA or ClpC results in the intracellular accumulation of the SigX in S. mutans. Together, our data support the notion that MecA mediates the formation of a ternary SigX-MecA-ClpC complex that sequesters SigX and thereby negatively regulates genetic competence in S. mutans. PMID:24039267

  14. 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

  15. RanBPM Protein Acts as a Negative Regulator of BLT2 Receptor to Attenuate BLT2-mediated Cell Motility*

    PubMed Central

    Wei, Jun-Dong; Kim, Joo-Young; Kim, Ae-Kyoung; Jang, Sung Key; Kim, Jae-Hong

    2013-01-01

    BLT2, a low affinity receptor for leukotriene B4 (LTB4), is a member of the G protein-coupled receptor family and is involved in many signal transduction pathways associated with various cellular phenotypes, including chemotactic motility. However, the regulatory mechanism for BLT2 has not yet been demonstrated. To understand the regulatory mechanism of BLT2, we screened and identified the proteins that bind to BLT2. Using a yeast two-hybrid assay with the BLT2 C-terminal domain as bait, we found that RanBPM, a previously proposed scaffold protein, interacts with BLT2. We demonstrated the specific interaction between BLT2 and RanBPM by GST pulldown assay and co-immunoprecipitation assay. To elucidate the biological function of the RanBPM-BLT2 interaction, we evaluated the effects of RanBPM overexpression or knockdown. We found that BLT2-mediated motility was severely attenuated by RanBPM overexpression and that knockdown of endogenous RanBPM by shRNA strongly promoted BLT2-mediated motility, suggesting a negative regulatory function of RanBPM toward BLT2. Furthermore, we observed that the addition of BLT2 ligands caused the dissociation of BLT2 and RanBPM, thus releasing the negative regulatory effect of RanBPM. Finally, we propose that Akt-induced BLT2 phosphorylation at residue Thr355, which occurs after the addition of BLT2 ligands, is a potential mechanism by which BLT2 dissociates from RanBPM, resulting in stimulation of BLT2 signaling. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2 signaling to attenuate BLT2-mediated cell motility. PMID:23928309

  16. A Gram-Negative Bacterial Secreted Protein Types Prediction Method Based on PSI-BLAST Profile

    PubMed Central

    2016-01-01

    Prediction of secreted protein types based solely on sequence data remains to be a challenging problem. In this study, we extract the long-range correlation information and linear correlation information from position-specific score matrix (PSSM). A total of 6800 features are extracted at 17 different gaps; then, 309 features are selected by a filter feature selection method based on the training set. To verify the performance of our method, jackknife and independent dataset tests are performed on the test set and the reported overall accuracies are 93.60% and 100%, respectively. Comparison of our results with the existing method shows that our method provides the favorable performance for secreted protein type prediction. PMID:27563663

  17. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination

    PubMed Central

    Yang, Hyun-Jeong; Vainshtein, Anna; Maik-Rachline, Galia; Peles, Elior

    2016-01-01

    While the formation of myelin by oligodendrocytes is critical for the function of the central nervous system, the molecular mechanism controlling oligodendrocyte differentiation remains largely unknown. Here we identify G protein-coupled receptor 37 (GPR37) as an inhibitor of late-stage oligodendrocyte differentiation and myelination. GPR37 is enriched in oligodendrocytes and its expression increases during their differentiation into myelin forming cells. Genetic deletion of Gpr37 does not affect the number of oligodendrocyte precursor cells, but results in precocious oligodendrocyte differentiation and hypermyelination. The inhibition of oligodendrocyte differentiation by GPR37 is mediated by suppression of an exchange protein activated by cAMP (EPAC)-dependent activation of Raf-MAPK-ERK1/2 module and nuclear translocation of ERK1/2. Our data suggest that GPR37 regulates central nervous system myelination by controlling the transition from early-differentiated to mature oligodendrocytes. PMID:26961174

  18. Negative Regulation of Interferon-induced Transmembrane Protein 3 by SET7-mediated Lysine Monomethylation*

    PubMed Central

    Shan, Zhao; Han, Qinglin; Nie, Jia; Cao, Xuezhi; Chen, Zuojia; Yin, Shuying; Gao, Yayi; Lin, Fang; Zhou, Xiaohui; Xu, Ke; Fan, Huimin; Qian, Zhikang; Sun, Bing; Zhong, Jin; Li, Bin; Tsun, Andy

    2013-01-01

    Although lysine methylation is classically known to regulate histone function, its role in modulating antiviral restriction factor activity remains uncharacterized. Interferon-induced transmembrane protein 3 (IFITM3) was found monomethylated on its lysine 88 residue (IFITM3-K88me1) to reduce its antiviral activity, mediated by the lysine methyltransferase SET7. Vesicular stomatitis virus and influenza A virus infection increased IFITM3-K88me1 levels by promoting the interaction between IFITM3 and SET7, suggesting that this pathway could be hijacked to support infection; conversely, IFN-α reduced IFITM3-K88me1 levels. These findings may have important implications in the design of therapeutics targeting protein methylation against infectious diseases. PMID:24129573

  19. Mesalazine negatively regulates CDC25A protein expression and promotes accumulation of colon cancer cells in S phase.

    PubMed

    Stolfi, Carmine; Fina, Daniele; Caruso, Roberta; Caprioli, Flavio; Fantini, Massimo Claudio; Rizzo, Angelamaria; Sarra, Massimiliano; Pallone, Francesco; Monteleone, Giovanni

    2008-06-01

    Regular consumption of mesalazine has been associated with a reduced risk of colorectal cancer (CRC) in patients with inflammatory bowel disease. The molecular mechanisms underlying the antineoplastic effect of 5-aminosalicylic acid remain, however, poorly characterized. In this study, we examined whether mesalazine affects cell cycle progression and analyzed specific checkpoint pathways in experimental models of CRC. Mesalazine inhibited the growth of HCT-116 and HT-29 cells, two CRC cell lines that express either a wild-type or mutated p53. Cell cycle analysis revealed that mesalazine induced cells to accumulate in S phase. This effect was associated with a sustained phosphorylation of the cyclin-dependent kinase (CDK)2 at threonine 14 and tyrosine 15 residues, an event that inactivates the CDK2-cyclin complex and blocks S-G(2) phase cell cycle transition. Consistently, mesalazine reduced the protein content of CDC25A, a phosphatase that regulates CDK2 phosphorylation status. Analysis of upstream kinases that negatively control CDC25A expression showed that mesalazine enhanced the activation of CHK1 and CHK2. However, silencing of CHK1 and CHK2 did not prevent the mesalazine-induced CDC25A protein downregulation. In contrast, CDC25A protein ubiquitination and degradation and accumulation of cells in S phase following mesalazine exposure were reverted by proteasome inhibitors. Notably, mesalazine also inhibited CDC25A in human CRC explants. Finally, we showed that mesalazine downregulated CDC25A in CT26, a murine CRC cell line, and prevented the formation of CT26-derived tumors in mice. Data show that mesalazine negatively regulates CDC25A protein expression, thus delaying CRC cell progression. PMID:18495657

  20. One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode.

    PubMed

    Kuo, Ting-Yu; Hong, Zhi-Wei; Tsai, Chung-Che; Yang, Yu-Chi; Fu, Hua-Wen

    2016-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy. PMID:27404433

  1. Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Huang, Yafan; Li, Hui; Hutchison, Claire E.; Laskey, James; Kieber, Joseph J.

    2003-01-01

    CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

  2. Molecular dynamic study of MlaC protein in Gram-negative bacteria: conformational flexibility, solvent effect and protein-phospholipid binding.

    PubMed

    Huang, Yu-Ming M; Miao, Yinglong; Munguia, Jason; Lin, Leo; Nizet, Victor; McCammon, J Andrew

    2016-08-01

    The composition of the outer membrane in Gram-negative bacteria is asymmetric, with the lipopolysaccharides found in the outer leaflet and phospholipids in the inner leaflet. The MlaC protein transfers phospholipids from the outer to inner membrane to maintain such lipid asymmetry in the Mla pathway. In this work, we have performed molecular dynamics simulations on apo and phospholipid-bound systems to study the dynamical properties of MlaC. Our simulations show that the phospholipid forms hydrophobic interactions with the protein. Residues surrounding the entrance of the binding site exhibit correlated motions to control the site opening and closing. Lipid binding leads to increase of the binding pocket volume and precludes entry of the water molecules. However, in the absence of the phospholipid, water molecules can freely move in and out of the binding site when the pocket is open. Dehydration occurs when the pocket closes. This study provides dynamic information of the MlaC protein and may facilitate the design of antibiotics against the Mla pathway of Gram-negative bacteria. PMID:27111825

  3. Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

    PubMed

    Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

    2015-01-01

    Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi. PMID:25830634

  4. CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

    PubMed

    Teerawatanasuk, N; Skalnik, D G; Carr, L G

    1999-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene. PMID:9886051

  5. The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein

    PubMed Central

    Hwang, Jungwon; Suh, Hyun-Woo; Jeon, Young Ho; Hwang, Eunha; Nguyen, Loi T.; Yeom, Jeonghun; Lee, Seung-Goo; Lee, Cheolju; Kim, Kyung Jin; Kang, Beom Sik; Jeong, Jin-Ok; Oh, Tae-Kwang; Choi, Inpyo; Lee, Jie-Oh; Kim, Myung Hee

    2014-01-01

    The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX–TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation. PMID:24389582

  6. 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. PMID:26434587

  7. Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism*

    PubMed Central

    Kakoi, Hironori; Maeda, Shingo; Shinohara, Naohiro; Matsuyama, Kanehiro; Imamura, Katsuyuki; Kawamura, Ichiro; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-01-01

    Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism. PMID:24505141

  8. Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3

    PubMed Central

    Rodrigues-Ferreira, Sylvie; Nehlig, Anne; Bouchet, Benjamin Pierre; Morel, Marina; Leconte, Ludovic; Serre, Laurence; Arnal, Isabelle; Braguer, Diane; Savina, Ariel; Honore, Stéphane; Nahmias, Clara

    2015-01-01

    The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor. PMID:26498358

  9. Negative regulation of EB1 turnover at microtubule plus ends by interaction with microtubule-associated protein ATIP3.

    PubMed

    Velot, Lauriane; Molina, Angie; Rodrigues-Ferreira, Sylvie; Nehlig, Anne; Bouchet, Benjamin Pierre; Morel, Marina; Leconte, Ludovic; Serre, Laurence; Arnal, Isabelle; Braguer, Diane; Savina, Ariel; Honore, Stéphane; Nahmias, Clara

    2015-12-22

    The regulation of microtubule dynamics is critical to ensure essential cell functions. End binding protein 1 (EB1) is a master regulator of microtubule dynamics that autonomously binds an extended GTP/GDP-Pi structure at growing microtubule ends and recruits regulatory proteins at this location. However, negative regulation of EB1 association with growing microtubule ends remains poorly understood. We show here that microtubule-associated tumor suppressor ATIP3 interacts with EB1 through direct binding of a non-canonical proline-rich motif. Results indicate that ATIP3 does not localize at growing microtubule ends and that in situ ATIP3-EB1 molecular complexes are mostly detected in the cytosol. We present evidence that a minimal EB1-interacting sequence of ATIP3 is both necessary and sufficient to prevent EB1 accumulation at growing microtubule ends in living cells and that EB1-interaction is involved in reducing cell polarity. By fluorescence recovery of EB1-GFP after photobleaching, we show that ATIP3 silencing accelerates EB1 turnover at microtubule ends with no modification of EB1 diffusion in the cytosol. We propose a novel mechanism by which ATIP3-EB1 interaction indirectly reduces the kinetics of EB1 exchange on its recognition site, thereby accounting for negative regulation of microtubule dynamic instability. Our findings provide a unique example of decreased EB1 turnover at growing microtubule ends by cytosolic interaction with a tumor suppressor. PMID:26498358

  10. The Ca(2+)-calmodulin-activated protein phosphatase calcineurin negatively regulates EGF receptor signaling in Drosophila development.

    PubMed Central

    Sullivan, Kathleen M C; Rubin, Gerald M

    2002-01-01

    Calcineurin is a Ca(2+)-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca(2+) signals into changes in cell function and development. We carried out a dominant modifier screen in the Drosophila eye using an activated form of the catalytic subunit to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. We demonstrated that a second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of our results, we propose that calcineurin, upon activation by Ca(2+)-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of sprouty and the GTPase-activating protein Gap1. PMID:12019233

  11. Anomalous Negative Fluorescence Anisotropy in Yellow Fluorescent Protein (YFP 10C): Quantitative Analysis of FRET in YFP Dimers

    PubMed Central

    Shi, Xinghua; Basran, Jaswir; Seward, Harriet E.; Childs, William; Bagshaw, Clive R.; Boxer, Steven G.

    2008-01-01

    YFP is widely used as a genetically-encoded fluorescent marker in biology. In the course of a comprehensive study of this protein, we observed an unusual, negative fluorescence anisotropy at pH 6.0 (McAnaney, T. B., Zeng, W., Doe, C. F. E., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X. H., Boxer, S. G., and Bagshaw, C. R. (2005), Biochemistry 44, 5510–5524). Here we report that the fluorescence anisotropy of YFP 10C depends on protein concentration in the low micromolar range that was not expected. We propose that the negative anisotropy is a result of unidirectional Förster resonance energy transfer (FRET) in a dimer of YFP, with the donor chromophore in the neutral form and the acceptor chromophore in the anionic form. This unusual mechanism is supported by studies of a monomeric YFP (A206K YFP) and transient-absorption spectroscopy of YFP 10C. A detailed analysis of the chromophore transition dipole moment direction is presented. The anisotropy and rate constant of this energy transfer are consistent with values produced by an analysis of the dimer structure observed in crystals. PMID:18027983

  12. Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

    PubMed Central

    Liu, Ying; Sun, Baocun; Liu, Tieju; Zhao, Xiulan; Wang, Xudong; Li, Yanlei; Meng, Jie; Gu, Qiang; Liu, Fang; Dong, Xueyi; Liu, Peimei; Sun, Ran; Zhao, Nan

    2016-01-01

    Tumor cell vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs) have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograft-murine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. PMID:27366084

  13. BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

    PubMed Central

    Weber, Kathrin; Harper, Nicholas; Schwabe, John; Cohen, Gerald M.

    2013-01-01

    Summary BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models. PMID:24120870

  14. Amer2 protein is a novel negative regulator of Wnt/β-catenin signaling involved in neuroectodermal patterning.

    PubMed

    Pfister, Astrid S; Tanneberger, Kristina; Schambony, Alexandra; Behrens, Jürgen

    2012-01-13

    Wnt/β-catenin signaling is negatively controlled by the adenomatous polyposis coli (APC) tumor suppressor, which induces proteasomal degradation of β-catenin as part of the β-catenin destruction complex. Amer2 (APC membrane recruitment 2; FAM123A) is a direct interaction partner of APC, related to the tumor suppressor Amer1/WTX, but its function in Wnt signaling is not known. Here, we show that Amer2 recruits APC to the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate lipids via lysine-rich motifs and that APC links β-catenin and the destruction complex components axin and conductin to Amer2. Knockdown of Amer2 increased Wnt target gene expression and reporter activity in cell lines, and overexpression reduced reporter activity, which required membrane association of Amer2. In Xenopus embryos, Amer2 is expressed mainly in the dorsal neuroectoderm and neural tissues. Down-regulation of Amer2 by specific morpholino oligonucleotides altered neuroectodermal patterning, which could be rescued by expression of a dominant-negative mutant of Lef1 that interferes with β-catenin-dependent transcription. Our data characterize Amer2 for the first time as a negative regulator of Wnt signaling both in cell lines and in vivo and define Amer proteins as a novel family of Wnt pathway regulators. PMID:22128170

  15. CXCR4 Protein Epitope Mimetic Antagonist POL5551 Disrupts Metastasis and Enhances Chemotherapy Effect in Triple-Negative Breast Cancer.

    PubMed

    Xiang, Jingyu; Hurchla, Michelle A; Fontana, Francesca; Su, Xinming; Amend, Sarah R; Esser, Alison K; Douglas, Garry J; Mudalagiriyappa, Chidananda; Luker, Kathryn E; Pluard, Timothy; Ademuyiwa, Foluso O; Romagnoli, Barbara; Tuffin, Gérald; Chevalier, Eric; Luker, Gary D; Bauer, Michael; Zimmermann, Johann; Aft, Rebecca L; Dembowsky, Klaus; Weilbaecher, Katherine N

    2015-11-01

    The SDF-1 receptor CXCR4 has been associated with early metastasis and poorer prognosis in breast cancers, especially the most aggressive triple-negative subtype. In line with previous reports, we found that tumoral CXCR4 expression in patients with locally advanced breast cancer was associated with increased metastases and rapid tumor progression. Moreover, high CXCR4 expression identified a group of bone marrow-disseminated tumor cells (DTC)-negative patients at high risk for metastasis and death. The protein epitope mimetic (PEM) POL5551, a novel CXCR4 antagonist, inhibited binding of SDF-1 to CXCR4, had no direct effects on tumor cell viability, but reduced migration of breast cancer cells in vitro. In two orthotopic models of triple-negative breast cancer, POL5551 had little inhibitory effect on primary tumor growth, but significantly reduced distant metastasis. When combined with eribulin, a chemotherapeutic microtubule inhibitor, POL5551 additively reduced metastasis and prolonged survival in mice after resection of the primary tumor compared with single-agent eribulin. Hypothesizing that POL5551 may mobilize tumor cells from their microenvironment and sensitize them to chemotherapy, we used a "chemotherapy framing" dosing strategy. When administered shortly before and after eribulin treatment, three doses of POL5551 with eribulin reduced bone and liver tumor burden more effectively than chemotherapy alone. These data suggest that sequenced administration of CXCR4 antagonists with cytotoxic chemotherapy synergize to reduce distant metastases. PMID:26269605

  16. Sp3 proteins negatively regulate beta myosin heavy chain gene expression during skeletal muscle inactivity.

    PubMed

    Tsika, Gretchen; Ji, Juan; Tsika, Richard

    2004-12-01

    In adult skeletal muscle, beta myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression is down-regulated in response to muscle inactivity. Little is known about the signaling pathways and transcription factors that mediate this important functional response. This study demonstrates that increased binding of Sp3 to GC-rich elements in the betaMyHC promoter is a critical event in down-regulation of betaMyHC gene expression under non-weight-bearing conditions. Conversely, binding of Sp3 to these elements decreased while Sp1 binding increased with nuclear extracts from plantaris muscle exposed to mechanical overload, a stimulus that increases betaMyHC gene expression. In addition, these experiments revealed the existence of an Sp4-DNA binding complex when using adult skeletal muscle nuclear extract was used but not when nuclear extracts from cultured myotubes were used. Sp3 proteins are competitive inhibitors of Sp1-mediated betaMyHC reporter gene transactivation in both Drosophila SL-2 and mouse C2C12 myotubes. Sp4 is a weak activator of betaMyHC gene expression in SL-2 cells, which lack endogenous Sp1 activity, but does not activate betaMyHC gene expression in C2C12 myotubes, which have high levels of Sp1. These results suggest that competitive binding of Sp family proteins regulate betaMyHC gene transcription in response to altered neuromuscular activity. PMID:15572681

  17. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability.

    PubMed

    Wu, Huixing; Kuzmenko, Alexander; Wan, Sijue; Schaffer, Lyndsay; Weiss, Alison; Fisher, James H; Kim, Kwang Sik; McCormack, Francis X

    2003-05-01

    The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A-null mice and was increased in SP-D-overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D-mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane. PMID:12750409

  18. Valosin-Containing Protein (VCP/p97)-Expression Correlates with Prognosis of HPV- Negative Oropharyngeal Squamous Cell Carcinoma (OSCC)

    PubMed Central

    Meyer, Moritz F.; Seuthe, Inga M. C.; Drebber, Uta; Siefer, Oliver; Kreppel, Matthias; Klein, Marcus O.; Mikolajczak, Stefanie; Klussmann, Jens Peter

    2014-01-01

    Valosin-containing protein (VCP)/p97 has been shown to be associated with antiapoptotic function via activation of the nuclear factor-B (NFB) signaling pathway and with metastasizing of tumors in several studies. VCP is located on chromosome 9p13-p12, a region often deleted in oropharyngeal squamous cell carcinoma (OSCC). The clinical significance of VCP expression in OSCC however remains unclear. In this study, expression of VCP was determined in 106 patients (77 male (71.3%) and 31 female (28.7%); age-range: 34–79 years (mean age 57 years)) by immunohistochemistry and in a subset of 15 patients by quantitative PCR. HPV-DNA was detected by polymerase chain reaction and p16INK4a immunohistochemistry. The experimental findings were correlated with clinico-pathological data and survival parameters. 47.2% of all OSCC specimens were analyzed as negative or weak staining intensity for VCP. 52.8% of all specimens showed a high staining intensity for VCP. 73.1% of all patients were tested HPV-negative, 26.9% were HPV-positive. The 5-year disease-free and overall survival probabilities of all patients were 71.2% and 55.7%, respectively. No correlation could be found between HPV-status and VCP expression. VCP overexpression in HPV-negative patients was associated with significantly better 5-year disease-free survival (86.4% vs., 45.6%, p = 0.017). The level of VCP-intensity determined by immunohistochemistry could be an additional prognostic marker in HPV-negative OSCC. VCP expression seems not to correlate with the HPV-status. PMID:25463965

  19. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli

    PubMed Central

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  20. Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with the Gram-Negative Bacterium Escherichia coli.

    PubMed

    Srivastava, Aashish; Asahara, Haruichi; Zhang, Meng; Zhang, Weijia; Liu, Haiying; Cui, Sheng; Jin, Qi; Chong, Shaorong

    2016-01-01

    Protein translation is essential for all bacteria pathogens. It has also been a major focus of structural and functional studies and an important target of antibiotics. Here we report our attempts to biochemically reconstitute mycobacterial protein translation in vitro from purified components. This mycobacterial translation system consists of individually purified recombinant translation factors from Mycobacterium tuberculosis (M. tuberculosis), purified tRNAs and ribosomes from Mycobacterium smegmatis (M. smegmatis), and an aminoacyl-tRNA synthetase (AARS) mixture from the cell-extract of M. smegmatis. We demonstrate that such mycobacterial translation system was efficient in in vitro protein synthesis, and enabled functional comparisons of translational components between the gram-positive Mycobacterium and the gram-negative E. coli. Although mycobacterial translation factors and ribosomes were highly compatible with their E. coli counterparts, M. smegmatis tRNAs were not properly charged by the E. coli AARSs to allow efficient translation of a reporter. In contrast, both E. coli and M. smegmatis tRNAs exhibited similar activity with the semi-purified M. smegmatis AARSs mixture for in vitro translation. We further demonstrated the use of both mycobacterial and E. coli translation systems as comparative in vitro assays for small-molecule antibiotics that target protein translation. While mycobacterial and E. coli translation were both inhibited at the same IC50 by the antibiotic spectinomycin, mycobacterial translation was preferentially inhibited by the antibiotic tetracycline, suggesting that there may be structural differences at the antibiotic binding sites between the ribosomes of Mycobacterium and E. coli. Our results illustrate an alternative approach for antibiotic discovery and functional studies of protein translation in mycobacteria and possibly other bacterial pathogens. PMID:27564552

  1. MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability

    PubMed Central

    Sun, Yang; Qin, Zhen; Li, Qi; Wan, Jing-jing; Cheng, Ming-he; Wang, Peng-yuan; Su, Ding-feng; Yu, Jian-guang; Liu, Xia

    2016-01-01

    Aim: MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. Methods: Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3′-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). Results: LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. Conclusion: miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases. PMID:27063215

  2. A Lotus japonicus Cochaperone Protein Interacts With the Ubiquitin-Like Domain Protein CIP73 and Plays a Negative Regulatory Role in Nodulation.

    PubMed

    Kang, Heng; Xiao, Aifang; Huang, Xiaoqin; Gao, Xioumei; Yu, Haixiang; He, Xingxing; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming

    2015-05-01

    The calcium/calmodulin-dependent protein kinase CCaMK forms a complex with its phosphorylation target CIP73 (CCaMK-interacting protein of 73 kDa). In this work, a homolog of the animal HSC/HSP70 interacting protein (HIP) was identified as an interacting partner of CIP73 in Lotus japonicus. L. japonicus HIP contains all functional domains characteristic of animal HIP proteins. The C-terminal STI1-like domain of L. japonicus HIP was found to be necessary and sufficient for interaction with CIP73. The interaction between CIP73 and HIP occurred in both the nuclei and cytoplasm in Nicotiana benthamiana leaf cells. The interactions between CIP73 and HIP and between CIP73 and CCaMK could take place simultaneously in the same nuclei. HIP transcripts were detected in all plant tissues tested. As nodule primordia developed into young nodules, the expression of HIP was down-regulated and the HIP transcript level became very low in mature nodules. More nodules were formed in transgenic hairy roots of L. japonicus expressing HIP RNA interference at 16 days postinoculation as compared with the control hairy roots expressing the empty vector. It appears that HIP may play a role as a negative regulator for nodulation. PMID:25761207

  3. Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling

    PubMed Central

    Holmes, Derek A.; Suto, Eric; Lee, Wyne P.; Ou, Qinglin; Gong, Qian; Smith, Hamish R.C.; Caplazi, Patrick

    2015-01-01

    The protein tyrosine phosphatase PTPN22(C1858T) allelic polymorphism is associated with increased susceptibility for development of systemic lupus erythematosus (SLE) and other autoimmune diseases. PTPN22 (also known as LYP) and its mouse orthologue PEP play important roles in antigen and Toll-like receptor signaling in immune cell functions. We demonstrate here that PEP also plays an important inhibitory role in interferon-α receptor (IFNAR) signaling in mice. PEP co-immunoprecipitates with components of the IFNAR signaling complex. Pep−/− hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep+/+ progenitors in response to IFN-α. In addition, Pep−/− mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α–treated Pep+/+ mice. As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias. PMID:26077719

  4. JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

    PubMed Central

    Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea

    2014-01-01

    JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. PMID:25332239

  5. The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis.

    PubMed

    Santner, Aaron A; Watson, John C

    2006-03-01

    The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants. PMID:16460509

  6. Polymorphism Analysis Reveals Reduced Negative Selection and Elevated Rate of Insertions and Deletions in Intrinsically Disordered Protein Regions

    PubMed Central

    Khan, Tahsin; Douglas, Gavin M.; Patel, Priyenbhai; Nguyen Ba, Alex N.; Moses, Alan M.

    2015-01-01

    Intrinsically disordered protein regions are abundant in eukaryotic proteins and lack stable tertiary structures and enzymatic functions. Previous studies of disordered region evolution based on interspecific alignments have revealed an increased propensity for indels and rapid rates of amino acid substitution. How disordered regions are maintained at high abundance in the proteome and across taxa, despite apparently weak evolutionary constraints, remains unclear. Here, we use single nucleotide and indel polymorphism data in yeast and human populations to survey the population variation within disordered regions. First, we show that single nucleotide polymorphisms in disordered regions are under weaker negative selection compared with more structured protein regions and have a higher proportion of neutral non-synonymous sites. We also confirm previous findings that nonframeshifting indels are much more abundant in disordered regions relative to structured regions. We find that the rate of nonframeshifting indel polymorphism in intrinsically disordered regions resembles that of noncoding DNA and pseudogenes, and that large indels segregate in disordered regions in the human population. Our survey of polymorphism confirms patterns of evolution in disordered regions inferred based on longer evolutionary comparisons. PMID:26047845

  7. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

    PubMed

    Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A; Barrett, Timothy G

    2015-02-01

    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis. PMID:25274773

  8. Peptidoglycan recognition protein-S5 functions as a negative regulator of the antimicrobial peptide pathway in the silkworm, Bombyx mori.

    PubMed

    Chen, Kangkang; Zhou, Lin; Chen, Feng; Peng, Yachun; Lu, Zhiqiang

    2016-08-01

    Prophenoloxidase (proPO), immune deficiency (IMD), and Toll are the major signaling pathways leading to melanization and antimicrobial peptide production in insect hemolymph. Peptidoglycan recognition proteins (PGRPs) act as receptors and negative regulators in these pathways, and some PGRPs exhibit antimicrobial activity. Previously, we demonstrated that silkworm PGRP-S5 recognizes peptidoglycans (PGs) and triggers activation of the proPO pathway. It also acts as a bactericide, via its amidase activity (Chen et al., 2014). Here, we generated a C177S site-mutated silkworm PGRP-S5 protein that lacked amidase activity but retained its PG-binding capacity. Functional studies showed that the mutation caused loss of its receptor function for activation of the proPO pathway, suggesting that processing of PG by PGRP-S5 is necessary for formation of the pathway initiation complex. Further, we found that PGRP-S5 negatively regulates antimicrobial peptides generation in an amidase-dependent manner, likely through the IMD pathway. Thus, silkworm PGRP-S5 acts as a sensor, a modulator, and an effector in the silkworm humoral immune system. PMID:27012996

  9. HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients.

    PubMed

    Kurozumi, Sasagu; Padilla, Mary; Kurosumi, Masafumi; Matsumoto, Hiroshi; Inoue, Kenichi; Horiguchi, Jun; Takeyoshi, Izumi; Oyama, Tetsunari; Ranger-Moore, Jim; Allred, D Craig; Dennis, Eslie; Nitta, Hiroaki

    2016-07-01

    HER2 gene-protein assay (GPA) is a new method for the simultaneous evaluation of HER2 immunohistochemistry (IHC) and HER2 dual in situ hybridization (DISH) on single tissue sections of breast cancer. We investigated the presence of HER2 gene and protein discrepancy and HER2-heterogeneity using HER2-GPA. HER2 status was analyzed for the correlation between the presence of HER2-heterogeneity and patient prognosis. Consecutive 280 invasive breast cancer were examined. Statuses of HER2 protein and gene were evaluated in whole tumor sections of HER2 GPA slides. HER2 protein and gene combination patterns were classified to six phenotypic and genotypic types for each case, as well as at individual cell levels: (A) IHC and DISH positive; (B) IHC positive and DISH negative; (C) IHC equivocal and DISH positive; (D) IHC equivocal and DISH negative; (E) IHC negative and DISH positive; and (F) IHC and DISH negative. The presence of HER2-heterogeneity was determined by the existence of at least two of six types within one tumor. HER2-IHC positive patients had significantly worse survival than IHC negative patients and HER2-DISH positive patients had significantly worse survival than DISH negative patients. HER2 IHC negative and DISH positive patients had significantly worse recurrence-free survival than IHC and DISH negative patients. In the HER2 IHC and DISH negative group, the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Notably, among triple negative breast cancer (TNBC), the HER2 heterogeneous group had significantly worse survival than the nonheterogeneous group. Our study suggests that the presence of HER2-heterogeneity might be a prognostic factor in HER2 negative breast cancer patients, especially in TNBC. PMID:27318853

  10. Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

    PubMed Central

    Liang, Zhe; Xie, Yan; Dominguez, Jessica A.; Breed, Elise R.; Yoseph, Benyam P.; Burd, Eileen M.; Farris, Alton B.

    2014-01-01

    Background Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Methods Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. Results In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Conclusions Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice. PMID:25010671

  11. Effectiveness of four dynamic treatment strategies in patients with anticitrullinated protein antibody-negative rheumatoid arthritis: a randomised trial

    PubMed Central

    Akdemir, G; Markusse, IM; Dirven, L; Riyazi, N; Steup-Beekman, GM; Kerstens, PJSM; Lems, WF; Huizinga, TWJ; Allaart, CF

    2016-01-01

    Objective To determine the most effective treatment strategy among anticitrullinated protein antibodies (ACPA)-negative patients with early rheumatoid arthritis. Methods In the BeSt study, 184 ACPA-negative patients were randomised to: (1) sequential monotherapy, (2) step-up therapy, (3) initial combination including prednisone, (4) initial combination including infliximab. Treatment was targeted at the disease activity score (DAS) ≤2.4. Early response and 10-year outcomes were compared between the four strategy-arms in ACPA-negative patients. Results ACPA-negative patients achieved more short-term functional improvement from initial combination therapy than when on monotherapy (at month 3, mean Health Assessment Questionnaire (HAQ) 0.71 vs 0.98, p=0.006; at month 6, 0.59 vs 0.87, p=0.004). Functional ability over time was comparable between the strategy-arms (p=0.551) with a mean HAQ of 0.6 at year 10 (p=0.580 for comparison across the strategy-arms). 10-year radiographic progression was negligible (median 0.5) and comparable between the 4 strategy-arms (p=0.082). At year 10, remission was achieved by 11/40 (28%), 9/45 (20%), 17/56 (30%) and 17/43 patients (40%) in strategy-arms 1–4, respectively (p=0.434). Over time, similar remission percentages were achieved in all strategy-arms (p=0.815). 18%, 16%, 20% and 21% in strategy-arms 1 to 4 (p=0.742) were in drug-free remission at year 10, with a median duration of 60 months across the arms. Conclusions Initial combination therapy with methotrexate, sulfasalazine and prednisone, or methotrexate and infliximab, is the most effective treatment strategy for ACPA-negative patients, resulting in earlier functional improvement than when on initial methotrexate monotherapy. After 10 years of targeted treatment, in all strategy-arms favourable clinical outcomes were achieved and radiographic progression was limited. Trial registration number NTR262, NTR265. PMID:27099776

  12. Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections.

    PubMed

    Wang, Xia; Du, Xiaoyuan; Li, Hongyan; Zhang, Shicui

    2016-02-19

    Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11-37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals. PMID:26740623

  13. A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis

    PubMed Central

    Chien, Pei-Shan; Nam, Hong Gil; Chen, Yet-Ran

    2015-01-01

    High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance. PMID:26093145

  14. Mir-190b negatively contributes to the Trypanosoma cruzi- infected cell survival by repressing PTEN protein expression

    PubMed Central

    Monteiro, Cíntia Júnia; Mota, Suianne Letícia Antunes; Diniz, Lívia de Figueiredo; Bahia, Maria Terezinha; Moraes, Karen CM

    2015-01-01

    Chagas disease, which is caused by the intracellular protozoanTrypanosoma cruzi, is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol-3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells. PMID:26692329

  15. Human T-cell lymphotropic virus type 3: complete nucleotide sequence and characterization of the human tax3 protein.

    PubMed

    Calattini, Sara; Chevalier, Sébastien Alain; Duprez, Renan; Afonso, Philippe; Froment, Alain; Gessain, Antoine; Mahieux, Renaud

    2006-10-01

    We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3Pyl43 provirus. As expected, HTLV-3Pyl43 contains open reading frames corresponding to the gag, pol, env, tax, and rex genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3Pyl43 is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3Pyl43 lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3Pyl43 displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3Pyl43 activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-kappaB pathway are similar but not identical in Tax1 and Tax3Pyl43. We also show that Tax3Pyl43 transactivates the human interleukin-8 and Bcl-XL promoters through the induction of the NF-kappaB pathway. On the other hand, Tax3Pyl43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3Pyl43 is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo. PMID:16973592

  16. RTP1 encodes a novel endoplasmic reticulum (ER)-localized protein in Arabidopsis and negatively regulates resistance against biotrophic pathogens.

    PubMed

    Pan, Qiaona; Cui, Beimi; Deng, Fengyan; Quan, Junli; Loake, Gary J; Shan, Weixing

    2016-03-01

    Oomycete pathogens cause serious damage to a wide spectrum of plants. Although host pathogen recognition via pathogen effectors and cognate plant resistance proteins is well established, the genetic basis of host factors that mediate plant susceptibility to oomycete pathogens is relatively unexplored. Here, we report on RTP1, a nodulin-related MtN21 family gene in Arabidopsis that mediates susceptibility to Phytophthora parasitica. RTP1 was identified by screening a T-DNA insertion mutant population and encoded an endoplasmic reticulum (ER)-localized protein. Overexpression of RTP1 rendered Arabidopsis more susceptible, whereas RNA silencing of RTP1 led to enhanced resistance to P. parasitica. Moreover, an RTP1 mutant, rtp1-1, displayed localized cell death, increased reactive oxygen species (ROS) production and accelerated PR1 expression, compared to the wild-type Col-0, in response to P. parasitica infection. rtp1-1 showed a similar disease response to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, including increased disease resistance, cell death and ROS production. Furthermore, rpt1-1 exhibited resistance to the fungal pathogen Golovinomyces cichoracearum, but not to the necrotrophic pathogen Botrytis cinerea. Taken together, these results suggest that RTP1 negatively regulates plant resistance to biotrophic pathogens, possibly by regulating ROS production, cell death progression and PR1 expression. PMID:26484750

  17. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  18. Negative superhelicity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA.

    PubMed

    Sung, P; Watkins, J F; Prakash, L; Prakash, S

    1994-03-18

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. Remarkable homology exists between RAD3 and the human excision repair gene XPD, whose mutational inactivation underlies the cancer-prone disorder in xeroderma pigmentosum group D patients. Our previous work demonstrated that RAD3-encoded protein contains a DNA helicase activity. Here, we show that RAD3 binds preferentially to UV-damaged DNA over nondamaged DNA. Removal of pyrimidine dimers from damaged DNA by enzymatic photoreactivation does not affect binding, suggesting an affinity of RAD3 for pyrimidine (6-4) pyrimidone photoproducts. Damage-specific binding by RAD3 is strongly dependent on ATP and on the degree of negative superhelicity in DNA. The requirement of superhelicity in damage binding may target RAD3 to regions of DNA undergoing transcription, resulting in the preferential repair of these regions. The rad3 Arg-48 mutant protein, which lacks the DNA helicase activity, also binds UV-damaged DNA preferentially, indicating that DNA helicase and damage binding are two distinct and separable functional entities in RAD3. PMID:8132553

  19. Negative regulation of microRNA-132 in expression of synaptic proteins in neuronal differentiation of embryonic neural stem cells.

    PubMed

    Yoshimura, Aya; Numakawa, Tadahiro; Odaka, Haruki; Adachi, Naoki; Tamai, Yoshitaka; Kunugi, Hiroshi

    2016-07-01

    MicroRNAs (miRs) play important roles in neuronal differentiation, maturation, and synaptic function in the central nervous system. They have also been suggested to be implicated in the pathogenesis of neurodegenerative and psychiatric diseases. Although miR-132 is one of the well-studied brain-specific miRs, which regulates synaptic structure and function in the postnatal brain, its function in the prenatal brain is still unclear. Here, we investigated miR-132 function during differentiation of rat embryonic neural stem cells (eNSCs). We found that miR-132 expression significantly increased during the fetal rat brain development and neural differentiation of eNSCs in vitro. Furthermore, miR-132 expression was increased during differentiation through MAPK/ERK1/2 pathway. Inhibition of ERK1/2 activation resulted in increased levels of synaptic proteins including PSD-95, GluR1 and synapsin I. Silencing of miR-132 also increased PSD-95 and GluR1. Considering that miR-132 increases synaptic proteins in differentiated cortical neurons, our result shows a novel function of miR-132 as a negative regulator for synaptic maturation in the neuronal differentiation of eNSCs. PMID:27131735

  20. A redundant nuclear protein binding site contributes to negative regulation of the mouse mammary tumor virus long terminal repeat.

    PubMed Central

    Bramblett, D; Hsu, C L; Lozano, M; Earnest, K; Fabritius, C; Dudley, J

    1995-01-01

    The tissue specificity of mouse mammary tumor virus (MMTV) expression is controlled by regulatory elements in the MMTV long terminal repeat (LTR). These regulatory elements include the hormone response element, located approximately between -200 and -75, as well as binding sites for NF-1, Oct-1 (OTF-1), and mammary gland enhancer factors. Naturally occurring MMTV deletion variants isolated from T-cell and kidney tumors, transgenic-mouse experiments with MMTV LTR deletions, and transient transfection assays with LTR constructs indicate that there are additional transcription regulatory elements, including a negative regulatory element (NRE), located upstream of the hormone response element. To further define this regulatory region, we have constructed a series of BAL 31 deletion mutants in the MMTV LTR for use in transient transfection assays. These assays indicated that deletion of two regions (referred to as promoter-distal and -proximal NREs) between -637 and -201 elevated basal MMTV promoter activity in the absence of glucocorticoids. The region between -637 and -264 was surveyed for the presence of nuclear protein binding sites by gel retardation assays. Only one type of protein complex (referred to as NRE-binding protein or NBP) bound exclusively to sites that mapped to the promoter-distal and -proximal NREs identified by BAL 31 mutations. The promoter-proximal binding site was mapped further by linker substitution mutations and transfection assays. Mutations that mapped to a region containing an inverted repeat beginning at -287 relative to the start of transcription elevated basal expression of a reporter gene driven by the MMTV LTR. A 59-bp DNA fragment from the distal NRE also bound the NBP complex. Gel retardation assays showed that mutations within both inverted repeats of the proximal NRE eliminated NBP binding and mutations within single repeats altered NBP binding. Intriguingly, the NBP complex was detected in extracts from T cells and lung cells but

  1. MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

    PubMed Central

    Mukhopadhyay, Partha; Lakshmanan, Imayavaramban; Ponnusamy, Moorthy P.; Chakraborty, Subhankar; Jain, Maneesh; Pai, Priya; Smith, Lynette M.; Lele, Subodh M.; Batra, Surinder K.

    2013-01-01

    Introduction Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs. Method In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining. Results MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue. Conclusions MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling. PMID

  2. The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond.

    PubMed

    Monaco, Giovanni; Vervliet, Tim; Akl, Haidar; Bultynck, Geert

    2013-04-01

    Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca(2+) signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca(2+) transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca(2+) oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca(2+)-channel tail and might be more crucial for Bcl-XL's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-XL's BH4 domains underpins their selective effect on Ca(2+) signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain's binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions. PMID:22955373

  3. Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice.

    PubMed

    Olenich, Sara A; Audet, Gerald N; Roberts, Kathleen A; Olfert, I Mark

    2014-08-01

    Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P < 0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix

  4. Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice

    PubMed Central

    Olenich, Sara A; Audet, Gerald N; Roberts, Kathleen A; Olfert, I Mark

    2014-01-01

    Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti-angiogenic peptides (thrombospondin-1, TSP-1; and endostatin) as well as pro-angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases-2 and -9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary-to-fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase-9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP-1 were elevated (P < 0.05). At D14 and D28, TSP-1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre-training levels. These data suggest that TSP-1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro-angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix

  5. Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

    PubMed

    Zhu, Wan Long; Tong, Honglian; Teh, Jing Tsong; Wang, Mei

    2014-01-01

    FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism. PMID:25546383

  6. Characterization of immune genes from the schistosome host snail Biomphalaria glabrata that encode peptidoglycan recognition proteins and gram-negative bacteria binding protein

    PubMed Central

    Zeng, Yong; Loker, Eric S.

    2013-01-01

    Peptidoglycan (PGN) recognition proteins (PGRPs) and gram-negative bacteria binding proteins (GNBPs) play an essential role in Toll/Imd signaling pathways in arthropods. The existence of homologous pathways involving PGRPs and GNBPs in other major invertebrate phyla such as the Mollusca remains unclear. In this paper, we report four full-length PGRP cDNAs and one full-length GNBP cDNA cloned from the snail Biomphalaria glabrata, the intermediate host of the human blood fluke Schistosoma mansoni, designated as BgPGRPs and BgGNBP, respectively. Three transcripts are generated from a long form PGRP gene (BgPGRP-LA) by alternative splicing and one from a short form PGRP gene (BgPGRP-SA). BgGNBP encodes a putative secreted protein. Northern blots demonstrated that expression of BgPGRP-SA and BgGNBP was down-regulated in B. glabrata at 6 h after exposure to three types of microbes. No significant changes in expression were observed in snails at 2 days post-exposure (dpe) to the trematodes Echinostoma paraensei or S. mansoni. However, up-regulation of BgPGRP-SA in M line snails at later time points of infection with E. paraensei (i.e., 12 and 17 dpe) was observed. Our study revealed that exposure to either microbes or trematodes did not alter the expression levels of BgPGRP-LAs, which were consistently low. This study provides new insights into the potential pathogen recognition capabilities of molluscs, indicates that further studies of the Toll/Imd pathways in this phylum are in order, and provides additional ways to judge the importance of this pathway in the evolution of internal defense across the animal phyla. PMID:17805526

  7. Electrospray mass spectrometry of some proteins and the aqueous solution acid/base equilibrium model in the negative ion detection mode

    NASA Astrophysics Data System (ADS)

    Le Blanc, J. C. Y.; Guevremont, R.; Siu, K. W. M.

    1993-06-01

    Basic solutions of myoglobin, [beta]-lactoglobulin, pepsin and ubiquitin have been examined by means of electrospray mass spectrometry in the negative ion detection mode. The distribution of protein ions in the mass spectra was found to correlate well with the distribution of protein species in solution calculated from published titration data. These results lend further credibility to an earlier proposed aqueous solution acid/base equilibrium model, which relates the "bellshape" ion distribution observed in the electrospray mass spectrometry of proteins to the distribution of protein ions in solution.

  8. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

    PubMed Central

    Jin, Benjamin Y.; Sartoretto, Juliano L.; Gladyshev, Vadim N.; Michel, Thomas

    2009-01-01

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells. PMID:19805165

  9. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells.

    PubMed

    Jin, Benjamin Y; Sartoretto, Juliano L; Gladyshev, Vadim N; Michel, Thomas

    2009-10-13

    Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells. PMID:19805165

  10. The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

    PubMed

    Carvalho, Raquel F; Szakonyi, Dóra; Simpson, Craig G; Barbosa, Inês C R; Brown, John W S; Baena-González, Elena; Duque, Paula

    2016-08-01

    The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars. PMID:27436712

  11. Insulin receptor substrate protein 53kDa (IRSp53) is a negative regulator of myogenic differentiation.

    PubMed

    Misra, Ashish; George, Bhawana; Rajmohan, Rajamuthiah; Jain, Neeraj; Wong, Ming Hwa; Kambadur, Ravi; Thanabalu, Thirumaran

    2012-06-01

    Fusion of mononucleated myoblasts to generate multinucleated myotubes is a critical step in skeletal muscle development. Filopodia, the actin cytoskeleton based membrane protrusions, have been observed early during myoblast fusion, indicating that they could play a direct role in myogenic differentiation. The control of filopodia formation in myoblasts remains poorly understood. Here we show that the expression of IRSp53 (Insulin Receptor Substrate protein 53kDa), a known regulator of filopodia formation, is down-regulated during differentiation of both mouse primary myoblasts and a mouse myoblast cell line C2C12. Over-expression of IRSp53 in C2C12 cells led to induction of filopodia and decrease in cell adhesion, concomitantly with inhibition of myogenic differentiation. In contrast, knocking down the IRSp53 expression in C2C12 cells led to a small but significant increase in myotube development. The decreased cell adhesion of C2C12 cells over-expressing IRSp53 is correlated with a reduction in the number of vinculin patches in these cells. Mutations in the conserved IMD domain (IRSp53 and MIM (missing in metastasis) homology domain) or SH3 domain of IRSp53 abolished the ability of this protein to inhibit myogenic differentiation and reduce cell adhesion. Over-expression of the IMD domain alone was sufficient to decrease the cell-extracellular matrix adhesion and to inhibit myogenesis in a manner dependent on its function in membrane shaping. Based on our data, we propose that IRSp53 is a negative regulator of myogenic differentiation which correlates with the observed down regulation of IRSp53 expression during myoblast differentiation to myotubes. PMID:22465711

  12. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors.

    PubMed

    Zhu, Yi; Tchkonia, Tamara; Fuhrmann-Stroissnigg, Heike; Dai, Haiming M; Ling, Yuanyuan Y; Stout, Michael B; Pirtskhalava, Tamar; Giorgadze, Nino; Johnson, Kurt O; Giles, Cory B; Wren, Jonathan D; Niedernhofer, Laura J; Robbins, Paul D; Kirkland, James L

    2016-06-01

    Clearing senescent cells extends healthspan in mice. Using a hypothesis-driven bioinformatics-based approach, we recently identified pro-survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro-survival regulators identified was Bcl-xl. Here, we tested whether the Bcl-2 family inhibitors, navitoclax (N) and TW-37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl-xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl-2, Bcl-xl, and Bcl-w, while T targets Bcl-2, Bcl-xl, and Mcl-1. The combination of Bcl-2, Bcl-xl, and Bcl-w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl-2, Bcl-xl, and Mcl-1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl-2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type-restricted manner. The hypothesis-driven, bioinformatics-based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type-specific senolytic agents. PMID:26711051

  13. Lipopolysaccharide-binding protein of Bombyx mori participates in a hemocyte-mediated defense reaction against gram-negative bacteria.

    PubMed

    Koizumi, N; Imai, Y; Morozumi, A; Imamura, M; Kadotani, T; Yaoi, K; Iwahana, H; Sato, R

    1999-09-01

    BmLBP is a lipopolysaccharide-binding protein in B. mori and participates in bacterial clearance in vivo. Here, we investigated the function of BmLBP more specifically. More than 90% of injected gram-negative rough strains to which BmLBP binds were removed from the plasma within 30 min post-injection, whereas it required 8h for the clearance of smooth strains to which BmLBP does not bind. Observation of the hemocoel after the injection of Escherichia coli rough strain showed that melanized nodules were formed at 30 min post-injection when the clearance of injected E. coli cells had occurred. Fluorescence microscope observation revealed that E. coli cells were actually trapped in the nodules formed in vivo. Furthermore, plasma pre-treated E. coli rough cells (BmLBP bound) added to hemocytes isolated in vitro caused vigorous hemocyte aggregations with the bacteria, while plasma pre-treated smooth cells did not. The formation of aggregates was inhibited by anti-BmLBP serum pre-treatment, suggesting that BmLBP causes the clearance of bacteria by promoting hemocyte nodule formation. PMID:12770298

  14. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria

    PubMed Central

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M.; Nichols, Wright W.

    2015-01-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  15. Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

    PubMed Central

    Boutry, Céline; Wahl, Astrid; Delplace, Brigitte; Clippe, André; Fontaine, Laetitia

    2012-01-01

    In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA (mecASt), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecASt represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus. PMID:22287513

  16. Distinctive Binding of Avibactam to Penicillin-Binding Proteins of Gram-Negative and Gram-Positive Bacteria.

    PubMed

    Asli, Abdelhamid; Brouillette, Eric; Krause, Kevin M; Nichols, Wright W; Malouin, François

    2016-02-01

    Avibactam is a novel non-β-lactam β-lactamase inhibitor that covalently acylates a variety of β-lactamases, causing inhibition. Although avibactam presents limited antibacterial activity, its acylation ability toward bacterial penicillin-binding proteins (PBPs) was investigated. Staphylococcus aureus was of particular interest due to the reported β-lactamase activity of PBP4. The binding of avibactam to PBPs was measured by adding increasing concentrations to membrane preparations of a variety of Gram-positive and Gram-negative bacteria prior to addition of the fluorescent reagent Bocillin FL. Relative binding (measured here as the 50% inhibitory concentration [IC50]) to PBPs was estimated by quantification of fluorescence after gel electrophoresis. Avibactam was found to selectively bind to some PBPs. In Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, and S. aureus, avibactam primarily bound to PBP2, with IC50s of 0.92, 1.1, 3.0, and 51 μg/ml, respectively, whereas binding to PBP3 was observed in Streptococcus pneumoniae (IC50, 8.1 μg/ml). Interestingly, avibactam was able to significantly enhance labeling of S. aureus PBP4 by Bocillin FL. In PBP competition assays with S. aureus, where avibactam was used at a fixed concentration in combination with varied amounts of ceftazidime, the apparent IC50 of ceftazidime was found to be very similar to that determined for ceftazidime when used alone. In conclusion, avibactam is able to covalently bind to some bacterial PBPs. Identification of those PBP targets may allow the development of new diazabicyclooctane derivatives with improved affinity for PBPs or new combination therapies that act on multiple PBP targets. PMID:26574008

  17. A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

    PubMed Central

    Steel, Gregor J.; Harley, Carol; Boyd, Alan; Morgan, Alan

    2000-01-01

    An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394 is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID:10749934

  18. Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  19. Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants.

    PubMed Central

    Szilvay, A M; Brokstad, K A; Kopperud, R; Haukenes, G; Kalland, K H

    1995-01-01

    The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype. PMID:7745679

  20. Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-XL.

    PubMed

    Wiesner, D A; Kilkus, J P; Gottschalk, A R; Quintáns, J; Dawson, G

    1997-04-11

    Prolonged (>24 h) exposure to anti-IgM (an antigen surrogate that induces membrane cross-linking and apoptosis) induced a 3-fold increase in the mass of endogenous ceramide measured by 32P labeling by diacylglycerol kinase and a 4-fold increase in ceramide as measured by metabolic labeling with [3H]palmitate in a B-lymphocyte cell line, WEHI 231. This correlated with the induction of apoptosis. Shorter exposure times to anti-IgM (up to 8 h) failed to elicit apoptosis and did not elicit increased ceramide formation. After 8 h, apoptosis occurs concomitantly with ceramide formation over the next 40 h. Further, we showed that exogenous ceramide mimicked anti-IgM-induced apoptosis and that apoptosis was potentiated in serum-free media. Treatment of cells with an inhibitor of ceramide catabolism, N-oleoylethanolamine, increased both ceramide formation and apoptosis and accelerated apoptosis induced by anti-IgM. To examine further how ceramide metabolism is involved in apoptosis, we derived cell lines from a small population of cells resistant to N-oleoylethanolamine. These cell lines were selected based on an altered ceramide metabolic pathway, were resistant to apoptosis induced by anti-IgM, and showed no significant increase in ceramide when challenged with anti-IgM. The basis of this resistance was shown to be the failure to activate neutral sphingomyelinase activity following 24-h treatment with anti-IgM, in contrast to the 2-fold increase in neutral sphingomyelinase activity observed in wild type cells. We have shown previously that transfection of WEHI cells with bcl-xL conferred resistance to anti-IgM-induced apoptosis, whereas transfection with bcl-2 did not (Gottschalk, A., Boise, L., Thompson, C., and Quintans, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7350-7354). In this study, these bcl-xL transfectants also displayed increased resistance to exogenous N-acetylsphingosine (C2-ceramide) or N-hexanoylsphingosine (C6-ceramide). However, when challenged with anti-IgM the bcl-xL transfectants produced levels of ceramide similar to wild type cells, suggesting that ceramide formation is upstream of bcl-xL and that it is a major determinant of B-cell death. PMID:9092523

  1. Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL

    SciTech Connect

    Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy

    2001-09-25

    Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. U sing isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells.

  2. Methylseleninic Acid Enhances Taxane Drug Efficacy against Human Prostate Cancer and Down-Regulates ntiapoptotic roteins Bcl-XL and Survivin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PURPOSE: Our previous work has shown that methylseleninic acid (MSeA) sensitized hormone refractory prostate cancer (HRPCa) cells to apoptosis induced by paclitaxel (taxol) through enhancing multiple caspases. This study aimed to: 1) determine the general applicability of the sensitization effect ...

  3. CHCHD2 connects mitochondrial metabolism to apoptosis

    PubMed Central

    Liu, Yong; Zhang, Yanping

    2015-01-01

    As the powerhouse of cells and gatekeeper for apoptosis, mitochondria control life and death. CHCHD2, a mitochondrial protein previously known to regulate metabolism, has recently been identified as an apoptosis inhibitor. New data suggest a model in which CHCHD2 performs a prosurvival function by acting as both a reactive oxygen species scavenger and BCL-XL activator. PMID:27308501

  4. HUMAN HOMOLOGUE OF S. POMBE RAD9 INTERACTS WITH BCL-2/BCL-X{SUB L} AND PROMOTES APOPTOSIS

    EPA Science Inventory

    DNA damage induces apoptosis through a Bcl-2 suppressible signaling pathway, but the mechanism is unknown. The human cell cycle checkpoint control protein HRad9 was found to interact with Bcl-2 and Bcl-XL but not Bax or Bad, as demonstrated by yeast two-hybrid and coimmunoprecipi...

  5. Breast Cancer Proteomics – Differences in Protein Expression between Estrogen Receptor-Positive and -Negative Tumors Identified by Tandem Mass Tag Technology

    PubMed Central

    Ruckhäberle, Eugen; Karn, Thomas; Hanker, Lars; Schwarz, Josef; Schulz-Knappe, Peter; Kuhn, Karsten; Böhm, Gitte; Selzer, Stefan; Erhard, Neukum; Engels, Knut; Holtrich, Uwe; Kaufmann, Manfred; Rody, Achim

    2010-01-01

    Background Proteomic analysis has become an effective tool in breast cancer research. In this study, we applied the new gel-free tandem mass tag (TMT) reference method for the first time in breast cancer. Materials and Methods Proteomic analysis was used to compare 10 estrogen receptor (ER)-positive and 10 ER-negative samples. The results of the proteomic approach were validated by Western blot, immunohistochemistry and gene expression analysis. Results 17 proteins with significant differences in expression were identified. 13 proteins were overexpressed in ER-negative tumors and 4 were overexpressed in ER-positive samples. All these proteins were characterized by relatively high cellular abundance. Conclusions Our results demonstrate that the gel-free TMT approach allows the quantification of differences in protein expression levels. Further improvement of the sensitivity by subfractionation of the tissue should allow also the identification of low-abundance proteins and might lead to the use of this method in breast cancer research. PMID:22619634

  6. Lactosylated PLGA nanoparticles containing ϵ-polylysine for the sustained release and liver-targeted delivery of the negatively charged proteins.

    PubMed

    Zhou, Ping; An, Tong; Zhao, Chuan; Li, Yuan; Li, Rongshan; Yang, Rui; Wang, Yinsong; Gao, Xiujun

    2015-01-30

    The acidic internal pH environment, initial burst release and lack of targeting property are main limitations of poly(lactide-co-glycolide) (PLGA) nanoparticles for carrying proteins. In this study, ϵ-polylysine (ϵ-PL) was used as an anti-acidic agent and a protein protectant to prepare PLGA nanoparticles for the protein delivery. To obtain the liver-targeting capability, lactosylated PLGA (Lac-PLGA) was synthesized by conjugation of lactose acid to PLGA at both ends, and then used to prepare nanoparticles containing ϵ-PL by the nanoprecipitation method. Bovine serumal bumin (BSA), a negatively charged protein, was efficiently loaded into Lac-PLGA/ϵ-PL nanoparticles and exhibited significant decreased burst release in vitro, sustained release in the blood and increased liver distribution in mice after intravenous injections. The enhanced stability of BSA was due to its electrical interaction with ϵ-PL and the neutralized internal environment of nanoparticles. In conclusion, Lac-PLGA/ϵ-PL nanoparticle system can be used as a promising carrier for the negatively charged proteins. PMID:25510599

  7. The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes

    PubMed Central

    Mercante, Virginia; Duarte, Cecilia M.; Sánchez, Cintia M.; Zalguizuri, Andrés; Caetano-Anollés, Gustavo; Lepek, Viviana C.

    2015-01-01

    Mesorhizobium loti MAFF303099 has a functional type III secretion system (T3SS) that is involved in the determination of nodulation competitiveness on Lotus. The M. loti T3SS cluster contains gene y4yS (mlr8765) that codes for a protein of unknown function (Y4yS). A mutation in the y4yS gene favors the M. loti symbiotic competitive ability on Lotus tenuis cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. In silico analysis indicated that this protein presents a tetratricopeptide repeat (TPR) domain, a signal peptide and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the y4yS-encoded protein is required for the formation of the M. loti T3SS secretin (RhcC2) complex. Remarkably, analysis of RhcC2 in the wild-type and M. loti y4yS mutant strains indicated that the absence of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane. PMID:25688250

  8. Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: Involvement of the effector proteins in the regulatory mechanism

    SciTech Connect

    Saxena, Sunil K. . E-mail: ssaxena@stevens.edu; Kaur, Simarna

    2006-07-21

    Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.

  9. A rare case of rapidly progressive dementia with elevated RT-QuIC and negative 14-3-3 and tau proteins.

    PubMed

    Trikamji, Bhavesh; Hamlin, Clive; Baldwin, Kelly J

    2016-05-01

    Creutzfeldt-Jakob disease (CJD) is characterized by rapidly progressing dementia with death usually occurring within 6 months. There is no verified disease-specific pre-mortem diagnostic test besides brain biopsy. We describe a 66 y old previously high functioning male who presented with a 5 month history of rapidly progressive dementia. Neurological examination revealed a score of 19/30 on MOCA testing. An extensive workup into various causes of dementia including electroencephalography and imaging studies was unremarkable. The cerebrospinal fluid was sent to National Prion Disease Center and it revealed elevated RT-QuIC levels with negative 14-3-3 and T tau proteins. Based on literature review, our case is one of few living subjects with elevated RT-QuIC levels and negative 14-3-3 and tau proteins. PMID:27249661

  10. Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling*

    PubMed Central

    Wang, Yu-Bo; Tan, Bo; Mu, Rui; Chang, Yan; Wu, Min; Tu, Hai-Qing; Zhang, Yu-Cheng; Guo, Sai-Sai; Qin, Xuan-He; Li, Tao; Li, Wei-Hua; Li, Ai-Ling; Zhang, Xue-Min; Li, Hui-Yan

    2015-01-01

    Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97. PMID:25681446